Antibodies to prostate-specific membrane antigen

ABSTRACT

This invention provides purified antibodies to the outer membrane domain of prostate-specific membrane (PSM) antigen, compositions of matter comprising PSM antigen antibodies conjugated to a radioisotope or a toxin, and a method of imaging prostate cancer by using PSM antigen antibodies.

This application is a continuation application of U.S. Ser. No.08/403,803, filed (Mar. 17, 1995, now abandoned and a continuation ofPCT International Application No. PCT/US93/10624, filed Nov. 5, 1993;which is a continuation-in-part of U.S. Ser. No. 07/973,337, filed Nov.5, 1992, now abandoned the contents of which are hereby incorporated byreference.

This invention disclosed herein was made in part with Government supportunder NIH Grants No. DK47650 and CA58192 from the Department of Healthand Human Services. Accordingly, the U.S. Government has certain rightsin this invention.

BACKGROUND OF THE INVENTION

Throughout this application various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofof each series of experiments.

Prostate cancer is among the most significant medical problems in theUnited States, as the disease is now the most common malignancydiagnosed in American males. In 1992 there were over 132,000 new casesof prostate cancer detected with over 36,000 deaths attributable to thedisease, representing a 17.3% increase over 4 years (2). Five yearsurvival rates for patients with prostate cancer range from 88% forthose with localized disease to 29% for those with metastatic disease.The rapid increase in the number of cases appears to result in part froman increase in disease awareness as well as the widespread use ofclinical markers such as the secreted proteins prostate-specific antigen(PSA) and prostatic acid phosphatase (PAP) (37).

The prostate gland is a site of significant pathology affected byconditions such as benign growth (BPH), neoplasia (prostatic cancer) andinfection (prostatitis). Prostate cancer represents the second leadingcause of death from cancer in man (1). However prostatic cancer is theleading site for cancer development in men. The difference between thesetwo facts relates to prostatic cancer occurring with increasingfrequency as men age, especially in the ages beyond 60 at a time whendeath from other factors often intervenes. Also, the spectrum ofbiologic aggressiveness of prostatic cancer is great, so that in somemen following detection the tumor remains a latent histologic tumor anddoes not become clinically significant, whereas in other it progressesrapidly, metastasizes and kills the man in a relatively short 2-5 yearperiod (1, 3).

In prostate cancer cells, two specific proteins that are made in veryhigh concentrations are prostatic acid phosphatase (PAP) and prostatespecific antigen (PSA) (4, 5, 6). These proteins have been characterizedand have been used to follow response to therapy. With the developmentof cancer, the normal architecture of the gland becomes altered,including loss of the normal duct structure for the removal ofsecretions and thus the secretions reach the serum. Indeed measurementof serum PSA is suggested as a potential screening method for prostaticcancer. Indeed, the relative amount of PSA and/or PAP in the cancerreduces as compared to normal or benign tissue.

PAP was one of the earliest serum markers for detecting metastaticspread (4). PAP hydrolyses tyrosine phosphate and has a broad substratespecificity. Tyrosine phosphorylation is often increased with oncogenictransformation. It has been hypothesized that during neoplastictransformation there is less phosphatase activity available toinactivate proteins that are activated by phosphorylation on tyrosineresidues. In some instances, insertion of phosphatases that havetyrosine phosphatase activity has reversed the malignant phenotype.

PSA is a protease and it is not readily appreciated how loss of itsactivity correlates with cancer development (5, 6). The proteolyticactivity of PSA is inhibited by zinc. Zinc concentrations are high inthe normal prostate and reduced in prostatic cancer. Possibly the lossof zinc allows for increased proteolytic activity by PSA. As proteasesare involved in metastasis and some proteases stimulate mitoticactivity, the potentially increased activity of PSA could behypothesized to play a role in the tumors metastases and spread (7).

Both PSA and PAP are found in prostatic secretions. Both appear to bedependent on the presence of androgens for their production and aresubstantially reduced following androgen deprivation.

Prostate-specific membrane antigen (PSM) which appears to be localizedto the prostatic membrane has been identified. This antigen wasidentified as the result of generating monoclonal antibodies to aprostatic cancer cell, LNCaP (8).

Dr. Horoszewicz established a cell line designated LNCaP from the lymphnode of a hormone refractory, heavily pretreated patient (9). This linewas found to have an aneuploid human male karyotype. It maintainedprostatic differentiation functionality in that it produced both PSA andPAP. It possessed an androgen receptor of high affinity and specificity.Mice were immunized with LNCaP cells and hybridomas were derived fromsensitized animals. A monoclonal antibody was derived and was designated7E11-C5 (8). The antibody staining was consistent with a membranelocation and isolated fractions of LNCaP cell membranes exhibited astrongly positive reaction with immunoblotting and ELISA techniques.This antibody did not inhibit or enhance the growth of LNCaP cells invitro or in vivo. The antibody to this antigen was remarkably specificto prostatic epithelial cells, as no reactivity was observed in anyother component. Immunohistochemical staining of cancerous epithelialcells was more intense than that of normal or benign epithelial cells.

Dr. Horoszewicz also reported detection of immunoreactive material using7E11-C5 in serum of prostatic cancer patients (8). The immunoreactivitywas detectable in nearly 60% of patients with stage D-2 disease and in aslightly lower percentage of patients with earlier stage disease, butthe numbers of patients in the latter group are small. Patients withbenign prostatic hyperplasia (BPH) were negative. Patients with noapparent disease were negative, but 50-60% of patients in remission yetwith active stable disease or with progression demonstrated positiveserum reactivity. Patients with non prostatic tumors did not showimmunoreactivity with 7E11-C5.

The 7E11-CS monoclonal antibody is currently in clinical trials. Thealdehyde groups of the antibody were oxidized and the linker-chelatorglycol-tyrosyl- (n, ε-diethylenetriamine-pentacetic acid)-lysine(GYK-DTPA) was coupled to the reactive aldehydes of the heavy chain(10). The resulting antibody was designated CYT-356. Immunohistochemicalstaining patterns were similar except that the CYT-356 modified antibodystained skeletal muscle. The comparison of CYT-356 with 7E11-CSmonoclonal antibody suggested both had binding to type 2 muscle fibers.The reason for the discrepancy with the earlier study, which reportedskeletal muscle to be negative, was suggested to be due to differencesin tissue fixation techniques. Still, the most intense and definitereaction was observed with prostatic epithelial cells, especiallycancerous cells. Reactivity with mouse skeletal muscle was detected withimmunohistochemistry but not in imaging studies. The Indium¹¹¹-labeledantibody localized to LNCaP tumors grown in nude mice with an uptake ofnearly 30% of the injected dose per gram tumor at four days. In-vivo, noselective retention of the antibody was observed in antigen negativetumors such as PC-3 and DU-145, or by skeletal muscle.

Very little was known about the PSM antigen. An effort at purificationand characterization has been described at meetings by Dr. George Wrightand colleagues (11, 12). These investigators have shown that followingelectrophoresis on acrylamide gels and Western blotting, the PSM antigenmaintains a molecular weight of 100 kilodaltons (kd). Chemical andenzymatic treatment showed that both the peptide and carbohydratemoieties of the PSM antigen are required for recognition by the 7E11-C5monoclonal antibody. Competitive binding studies with specific lectinssuggested that galNAc is the dominant carbohydrate of the antigenicepitope.

The 100 kd glycoprotein unique to prostate cells and tissues waspurified and characterized. The protein was digested proteolyticallywith trypsin and nine peptide fragments were sequenced. Using thetechnique of degenerate PCR (polymerase chain reaction), the full-length2.65 kilobase (kb) cDNA coding for this antigen was cloned. Preliminaryresults have revealed that this antigen is highly expressed in prostatecancer tissues, including bone and lymph node metastases (13). Theentire DNA sequence for the cDNA as well as the predicted amino acidsequence for the antigen was determined. Further characterization of thePSM antigen is presently underway in the applicants' laboratoryincluding: analysis of PSM gene expression in a wide variety of tissues,transfection of the PSM gene into cells not expressing the antigen,chromosome localization of the PSM gene, cloning of the genomic PSM genewith analysis of the PSM promoter and generation of polyclonal andmonoclonal antibodies against highly antigenic peptide domains of thePSM antigen, and identification of any endogenous PSM binding molecules(ligands).

Currently, LNCaP cells provide the best in-vitro model system to studyhuman prostate cancer, since they produce all three prostaticbio-markers; PSA, PAP and PSM. The cells possess an aneuploid malekaryotype with a Y chromosome, express a high affinity androgenreceptor, and are hormonally responsive to both testosterone and DHT.Because PSM appears to be a transmembrane glycoprotein, it is consideredan attractive target for both antibody-directed imaging and targeting ofprostatic tumor deposits (38). We have demonstrated expression of PSMprotein in LNCAP cell membranes and in PC-3 cells transfected with PSMcDNA and also the characterization of PSM mRNA expression in humantissues, and in response to steroid hormones.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Signal in lane 2 represent the 100 kD PSM antigen. The EGFr wasused as the positive control and is shown in lane 1. Incubation withrabbit antimouse (RAM) antibody alone served as negative control and isshown in lane 3.

FIG. 2 A-D: Upper two photos show LNCaP cytospins staining positivelyfor PSM antigen. Lower left in DU-145 and lower right is PC-3 cytospin,both negative for PSM antigen expression.

FIG. 3 A-D: Upper two panels are human prostate sections (BPH) stainingpositively for PSM antigen. The lower two panels show invasive prostatecarcinoma human sections staining positively for expression of the PSMantigen.

FIG. 4: 100 kD PSM antigen following immunoprecipitation of³⁵S-Methionine labelled LNCaP cells with Cyt-356 antibody.

FIG. 5: 3% agarose gels stained with Ethidium bromide revealing PCRproducts obtained using the degenerate PSM antigen primers. The arrowpoints to sample IN-20, which is a 1.1 kb PCR product which we laterconfirmed to be a partial cDNA coding for the PSM gene.

FIG. 6 A-B: 2% agarose gels of plasmid DNA resulting from TA cloning ofPCR products. Inserts are excised from the PCR II vector (InvitrogenCorp.) by digestion with EcoRI. 1.1 kb PSM gene partial cDNA product isshown in lane 3 of gel 1.

FIG. 7: Autoradiogram showing size of cDNA represented in applicants'LNCaP library using M-MLV reverse transcriptase.

FIG. 8: Restriction analysis of full-length clones of PSM gene obtainedafter screening cDNA library. Samples have been cut with Not I and Sal Irestriction enzymes to liberate the insert.

FIG. 9: Plasmid Southern autoradiogram of full length PSM gene clones.Size is approximately 2.7 kb.

FIG. 10: Northern blot revealing PSM expression limited to LNCaPprostate cancer line and H26 Ras-transfected LNCaP cell line. PC-3,DU-145, T-24, SKRC-27, HELA, MCF-7, HL-60, and others were are allnegative.

FIG. 11: Autoradiogram of Northern analysis revealing expression of 2.8kb PSM message unique to the LNCaP cell line (lane 1), and absent fromthe DU-145 (lane 2) and PC-3 cell lines (lane 3). RNA size ladder isshown on the left (kb), and 28S and 18S ribosomal RNA bands areindicated on the right.

FIG. 12 A-B: Results of PCR of human prostate tissues using PSM geneprimers. Lanes are numbered from left to right. Lane 1, LNCaP; Lane 2,H26; Lane 3, DU-145; Lane 4, Normal Prostate; Lane 5, BPH; Lane 6,Prostate Cancer; Lane 7, BPH; Lane 8, Normal; Lane 9, BPH; Lane 10, BPH;Lane 11, BPH; Lane 12, Normal; Lane 13, Normal; Lane 14, Cancer; Lane15, Cancer; Lane 16, Cancer, Lane 17, Normal; Lane 13, Cancer; Lane 19,IN-20 Control; Lane 20, PSM cDNA

FIG. 13: Isoelectric point of PSM antigen (non-glycosylated)

FIG. 14; 1-8 Secondary structure of antigen (panels 14-4 to 14-H: SEQ IDNO:2)

FIG. 15:A-B A. Hydrophilicity plot of PSM antigen B. Prediction ofmembrane spanning segments (SEQ ID NOS: 35-37).

FIG. 16:1-11 Homology of PSMA antigen (SEQ ID NO:1) with chicken (SEQ IDNO:27), rat (SEQ ID NO:28) and human (SEQ ID NO:29) transferrin receptorsequence.

FIG. 17A-C: Immunohistochemical detection of PSM antigen expression inprostate cell lines. Top panel reveals uniformly high level ofexpression in LNCaP cells; middle panel and lower panel are DU-145 andPC-3 cells respectively, both negative.

FIG. 18: Autoradiogram of protein gel revealing products of PSM coupledin-vitro transcription/translation. Non-glycosylated PSM polypeptide isseen at 84 kDa (lane 1) and PSM glycoprotein synthesized following theaddition of microsomes is seen at 100 kDa (lane 2).

FIG. 19: Western Blot analysis detecting PSM expression in transfectednon-PSM expressing PC-3 cells. 100 kDa PSM glycoprotein species isclearly seen in LNCaP membranes (lane 1), LNCaP crude lysate (lane 2),and PSM-transfected PC-3 cells (lane 4), but is undetectable in nativePC-3 cells (lane 3).

FIG. 20: Autoradiogram of ribonuclease protection gel assaying for PSMmRNA expression in normal human tissues. Radiolabeled 1 kb DNA ladder(Gibco-BRL) is shown in lane 1. Undigested probe is 400 nucleotides(lane 2), expected protected PSM band-is 350 nucleotides, and tRNAcontrol is shown (lane 3). A strong signal is seen in human prostate(lane 11), with very faint, but detectable signals seen in human brain(lane 4) and human salivary gland (lane 12).

FIG. 21: Autoradiogram of ribonuclease protection gel assaying for PSMmRNA expression in LNCaP tumors grown in nude mice, and in humanprostatic tissues. ³²P-labeled 1 kb DNA ladder is shown in lane 1. 298nucleotide undigested probe is shown (lane 2), and tRNA control is shown(lane 3). PSM mRNA expression is clearly detectable in LNCaP cells (lane4), orthotopically grown LNCaP tumors in nude mice with and withoutmatrigel (lanes 5 and 6), and subcutaneously implanted and grown LNCaPtumors in nude mice (lane 7). PSM mRNA expression is also seen in normalhuman prostate (lane 8), and in a moderately differentiated humanprostatic adenocarcinoma (lane 10). Very faint expression is seen in asample of human prostate tissue with benign hyperplasia (lane 9).

FIG. 22: Ribonuclease protection assay for PSM expression in LNCaP cellstreated with physiologic doses of various steroids for 24 hours.³²-Plabeled DNA ladder is shown in lane 1. 298 nucleotide undigestedprobe is shown (lane 2), and tRNA control is shown (lane 3). PSM mRNAexpression is highest in untreated LNCaP cells in charcoal-strippedmedia (lane 4). Applicant see significantly diminished PSM expression inLNCaP cells treated with DHT (lane 5) Testosterone (lane 6), Estradiol(lane 7), and Progesterone (lane 8), with little response toDexamethasone (lane 9).

FIG. 23: Data illustrating results of PSM DNA and RNA presence intransfect Dunning cell lines employing Southern and Northern blottingtechniques

FIG. 24:A-B Figure A indicates the power of cytokine transfected cellsto teach unmodified cells. Administration was directed to the parentalflank or prostate cells. The results indicate the microenvironmentconsiderations. Figure B indicates actual potency at a particular site.The tumor was implanted in prostate cells and treated with immune cellsat two different sites.

FIG. 25:A-B Relates potency of cytokines in inhibiting growth of primarytumors. Animals administered un-modified parental tumor cells andadministered as a vaccine transfected cells. Following prostatectomy ofrodent tumor results in survival increase.

FIG. 26: PCR amplification with nested primers improved our level ofdetection of prostatic cells from approximately one prostatic cell per10,000 MCF-7 cells to better than one cell per million MCF-7 cells,using either PSA.

FIG. 27: PCR amplification with nested primers improved our level ofdetection of prostatic cells from approximately one prostatic cell per10,000 MCF-7 cells to better than one cell per million MCF-7 cells,using PSM-derived primers.

FIG. 28: A representative ethidium stained gel photograph for PSM-PCR.Samples run in lane A represent PCR products generated from the outerprimers and samples in lanes labeled B are products of inner primerpairs.

FIG. 29: PSM Southern blot autoradiograph. The sensitivity of theSouthern blot analysis exceeded that of ethidium staining, as can beseen in several samples where the outer product is not visible on FIG. 3A-D, but is detectable by Southern blotting as shown in FIG. 4.

FIG. 30: Characteristics of the 16 patients analyzed with respect totheir clinical stage, treatment, serum PSA and PAP values, and resultsof assay.

SUMMARY OF THE INVENTION

This invention provides an isolated mammalian nucleic acid moleculeencoding a mammalian prostate-specific membrane (PSM) antigen. Theisolated mammalian nucleic acid may be DNA, cDNA or RNA.

This invention also provides nucleic acid molecule comprising a nucleicacid molecule of at least 15 nucleotides capable of specificallyhybridizing with a sequence included within the sequence of a nucleicacid molecule encoding the PSM antigen. The nucleic acid moleculemay-either be DNA or RNA.

This invention provides nucleic acid molecule of at least 15 nucleotidescapable of specifically hybridizing with a sequence of a nucleic acidmolecule which is complementary to the nucleic acid molecule encoding amammalian prostate-specific membrane antigen.

This invention further provides a method of detecting expression of thePSM antigen which comprises obtaining total mRNA from the cell andcontacting the mRNA so obtained with a labelled PSM antigen specificnucleic acid molecule under hybridizing conditions, determining thepresence of mRNA hybridized to the probe, and thereby detecting theexpression of the PSM antigen by the cell. The PSM antigen in tissuesections may be similarly detected.

This invention provides isolated nucleic acid molecule of PSM antigenoperatively linked to a promoter of RNA transcription. This inventionfurther provides a vector which comprises an isolated mammalian nucleicacid molecule of PSM antigen.

This invention further provides a host vector system for the productionof a polypeptide having the biological activity of a mammalian PSMantigen which comprises the vector comprising the mammalian nucleic acidmolecule encoding a mammalian PSM antigen and a suitable host. Thesuitable host for the expression of PSM antigen may be a bacterial cell,insect cell, or mammalian cell.

This invention also provides a method of producing a polypeptide havingthe biological activity of a mammalian PSM antigen which comprisesgrowing the host cell of vector system having a vector comprising theisolated mammalian nucleic acid molecule encoding a mammalian PSMantigen and a suitable host under suitable conditions permittingproduction of the polypeptide and recovery of the polypeptide soproduced.

This invention provides a method for determining whether a ligand canbind to a mammalian PSM antigen which comprises contacting a mammaliancell having an isolated mammalian DNA molecule encoding a mammalian PSMantigen with the ligand under conditions permitting binding of ligandsto the mammalian PSM antigen, and determining whether the ligand bindsto a mammalian PSM antigen. This invention further provides ligandswhich bind to PSM antigen.

This invention provides purified mammalian PSM antigen. This inventionalso provides a polypeptide encoded by the isolated mammalian nucleicacid molecule encoding a mammalian PSM antigen. This invention furtherprovides a method to identify and purify ligands of mammalian PSMantigen.

This invention further provides a method to produce both polyclonal andmonoclonal antibody using purified PSM antigens or polypeptides encodedby an isolated mammalian nucleic acid molecule encoding a mammalian PSMantigen.

This invention provides polyclonal and monoclonal antibody most likelybut not limited to directed either to peptide Asp-Glu-Leu-Lys-Ala-Glu(SEQ ID No. 35), or Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No. 36) orLys-Ser-Pro-Asp-Glu-Gly (SEQ ID No. 37) of the PSM antigen.

This invention provides a therapeutic agent comprising an antibodydirected against a mammalian PSM antigen and a cytotoxic agentconjugated thereto.

This invention also provides a method of imaging prostate cancer inhuman patients which comprises administering to the patient at least oneantibody directed against PSM antigen, capable of binding to the cellsurface of the prostate cancer cell and labeled with an imaging agentunder conditions so as to form a complex between the monoclonal antibodyand the cell surface PSM antigen. This invention further provides acomposition comprising an effective imaging amount of the antibodydirected against PSM antigen and a pharmaceutically acceptable carrier.

This invention further provides a method of imaging prostate cancer inhuman patients which comprises administering to the patient multipleantibodies directed towards different PSM epitopes.

The invention also provides a method of imaging prostate cancer in humanpatients which comprises administering to the patient at least oneligand, capable of binding to the cell surface of the prostate cancercell and labelled with an imaging agent under conditions so as to form acomplex between the ligand and the cell surface PSM antigen. Thisinvention further provides a composition comprising an effective imagingamount of PSM antigen and a pharmaceutically acceptable carrier.

This invention provides an immunoassay for measuring the amount of thePSM antigen in a biological sample, e.g. serum, comprising steps of a)contacting the biological sample with at least one PSM antibody to forma complex with said antibody and the PSM antigen, and b) measuring theamount of PSM antigen in said biological sample by measuring the amountof said complex.

This invention also provides an immunoassay for measuring the amount ofthe PSM antigen in a biological sample comprising steps of a) contactingthe biological sample with at least one PSM ligand to form a complexwith said ligand and the PSM antigen, and b) measuring the amount of thePSM antigen in said biological sample by measuring the amount of saidcomplex.

This invention provides a method to purify mammalian PSM antigencomprising steps of a) coupling the antibody directed against PSMantigen to a solid matrix; b) incubating the coupled antibody of a) witha cell lysate containing PSM antigen under the condition permittingbinding of the antibody and PSM antigen c) washing the coupled solidmatrix to eliminate impurities and d) eluting the PSM antigen from thebound antibody.

This invention further provides transgenic nonhuman mammals whichcomprises an isolated nucleic acid molecule of PSM antigen. Thisinvention also provides a transgenic nonhuman mammal whose genomecomprises antisense DNA complementary to DNA encoding a mammalian PSMantigen so placed as to be transcribed into antisense mRNA complementaryto mRNA encoding the PSM antigen and which hybridizes to mRNA encodingthe PSM antigen thereby reducing its translation.

This invention provides a method of suppressing or modulating metastaticability of prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells comprising introducing a DNA molecule encoding aprostate specific membrane antigen operatively linked to a 5′ regulatoryelement into a tumor cell of a subject, in a way that expression of theprostate specific membrane antigen is under the control of theregulatory element, thereby suppressing or modulating metastatic abilityof prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells.

This invention provides a method of suppressing or modulating metastaticability of prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells, comprising introducing a DNA molecule encoding aprostate specific membrane antigen operatively linked to a 5′ regulatoryelement coupled with a therapeutic DNA into a tumor cell of a subject,thereby suppressing or modulating metastatic ability of prostate tumorcells, prostate tumor growth or elimination of prostate tumor cells.

This invention provides a therapeutic vaccine for preventing humanprostate tumor growth or stimulation of prostate tumor cells in asubject, comprising administering an effective amount to the prostatecell, and a pharmaceutical acceptable carrier, thereby preventing thetumor growth or stimulation of tumor cells in the subject.

This invention provides a method of detecting hematogenous micrometastictumor cells of a subject, comprising (A) performing nested polymerasechain reaction (PCR) on blood, bone marrow, or lymph node samples of thesubject using the prostate specific membrane antigen primers, and (B)verifying micrometastases by DNA sequencing and Southern analysis,thereby detecting hematogenous micrometastic tumor cells of the subject.

This invention provides a method of abrogating the mitogenic responsedue to transferrin, comprising introducing a DNA molecule encodingprostate specific membrane antigen operatively linked to a 5′ regulatoryelement into a tumor cell, the expression of which gene is directlyassociated with a defined pathological effect within a multicellularorganism, thereby abrogating mitogen response due to transferrin.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, references to specific nucleotides are tonucleotides present on the coding strand of the nucleic acid. Thefollowing standard abbreviations are used throughout the specificationto indicate specific nucleotides:

-   -   C=cytosine A=adenosine    -   T=thymidine G=guanosine        A “gene” means a nucleic acid molecule, the sequence of which        includes all the information required for the normal regulated        production of a particular protein, including the structural        coding sequence, promoters and enhancers.

This invention provides an isolated mammalian nucleic acid encoding amammalian prostate-specific membrane (PSM) antigen.

This invention further provides an isolated mammalian DNA molecule of anisolated mammalian nucleic acid molecule encoding a mammalianprostate-specific membrane antigen. This invention also provides anisolated mammalian cDNA molecule encoding a mammalian prostate-specificmembrane antigen. This invention provides an isolated mammalian RNAmolecule encoding a mammalian prostate-specific membrane antigen.

In the preferred embodiment of this invention, the isolated nucleicsequence is cDNA from human as shown in sequence ID number 1. This humansequence was submitted to-GenBank (Los Alamos National Laboratory, LosAlamos, N.M.) with Accession Number, M99487 and the description as PSM,Homo sapiens, 2653 base-pairs.

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of PSM antigen, but which should notproduce phenotypic changes. Alternatively, this invention alsoencompasses DNAs and cDNAs which hybridize to the DNA and cDNA of thesubject invention. Hybridization methods are well known to those ofskill in the art.

The DNA molecules of the subject invention also include DNA moleculescoding for polypeptide analogs, fragments or derivatives of antigenicpolypeptides which differ from naturally-occurring forms in terms of theidentity or location of one or more amino acid residues (deletionanalogs containing less than all of the residues specified for theprotein, substitution analogs wherein one or more residues specified arereplaced by other residues and addition analogs where in one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors.

The DNA molecules described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The molecule isuseful for generating new cloning and expression vectors, transformedand transfected prokaryotic and eukaryotic host cells, and new anduseful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

Moreover, the isolated mammalian nucleic acid molecules encoding amammalian prostate-specific membrane antigen are useful for thedevelopment of probes to study the tumorigenesis of prostate cancer.

This invention also provides nucleic acid molecules of at least 15nucleotides capable of specifically hybridizing with a sequence of anucleic acid molecule encoding the prostate-specific membrane antigen.

This nucleic acid molecule produced can either be DNA or RNA. As usedherein, the phrase “specifically hybridizing” means the ability of anucleic acid molecule to recognize a nucleic acid sequence complementaryto its own and to form double-helical segments through hydrogen bondingbetween complementary base pairs.

This nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a sequence of a nucleic acid moleculeencoding the prostate-specific membrane antigen can be used as a probe.Nucleic acid probe technology is well known to those skilled in the artwho will readily appreciate that such probes may vary greatly in lengthand may be labeled with a detectable label, such as a radioisotope orfluorescent dye, to facilitate detection of the probe. DNA probemolecules may be produced by insertion of a DNA molecule which encodesPSM antigen into suitable vectors, such as plasmids or bacteriophages,followed by transforming into suitable bacterial host cells, replicationin the transformed bacterial host cells and harvesting of the DNAprobes, using methods well known in the art. Alternatively, probes maybe generated chemically from DNA synthesizers.

RNA probes may be generated by inserting the PSM antigen moleculedownstream of a bacteriophage promoter such as T3, T7 or SP6. Largeamounts of RNA probe may be produced by incubating the labelednucleotides with the linearized PSM antigen fragment where it containsan upstream promoter in the presence of the appropriate RNA polymerase.

This invention also provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of anucleic acid molecule which is complementary to the mammalian nucleicacid molecule encoding a mammalian prostate-specific membrane antigen.This molecule may either be a DNA or RNA molecule.

The current invention further provides a method of detecting theexpression of a mammalian PSM antigen expression in a cell whichcomprises obtaining total mRNA from the cell, contacting the mRNA soobtained with a labelled nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of thenucleic acid molecule encoding a mammalian PSM antigen under hybridizingconditions, determining the presence of mRNA hybridized to the moleculeand thereby detecting the expression of the mammalian prostate-specificmembrane antigen in the cell. The nucleic acid molecules synthesizedabove may be used to detect expression of a PSM antigen by detecting thepresence of mRNA coding for the PSM antigen. Total mRNA from the cellmay be isolated by many procedures well known to a person of ordinaryskill in the art. The hybridizing conditions of the labelled nucleicacid molecules may be determined by routine experimentation well knownin the art. The presence of mRNA hybridized to the probe may bedetermined by gel electrophoresis or other methods known in the art. Bymeasuring the amount of the hybrid made, the expression of the PSMantigen by the cell can be determined. The labelling may be radioactive.For an example, one or more radioactive nucleotides can be incorporatedin the nucleic acid when it is made.

In one embodiment of this invention, nucleic acids are extracted byprecipitation from lysed cells and the mRNA is isolated from the extractusing an oligo-dT column which binds the poly-A tails of the mRNAmolecules (13). The mRNA is then exposed to radioactively labelled probeon a nitrocellulose membrane, and the probe hybridizes to and therebylabels complementary mRNA sequences. Binding may be detected byluminescence autoradiography or scintillation counting. However, othermethods for performing these steps are well known to those skilled inthe art, and the discussion above is merely an example.

This invention further provides another method to detect expression of aPSM antigen in tissue sections which comprises contacting the tissuesections with a labelled nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence ofnucleic acid molecules encoding a mammalian PSM antigen underhybridizing conditions, determining the presence of mRNA hybridized tothe molecule and thereby detecting the expression of the mammalian PSMantigen in tissue sections. The probes are also useful for in-situhybridization or in order to locate tissues which express this gene, orfor other hybridization assays for the presence of this gene or its mRNAin various biological tissues. The in-situ hybridization using alabelled nucleic acid molecule is well known in the art. Essentially,tissue sections are incubated with the labelled nucleic acid molecule toallow the hybridization to occur. The molecule will carry a marker forthe detection because it is “labelled”, the amount of the hybrid will bedetermined based on the detection of the amount of the marker and sowill the expression of PSM antigen.

This invention further provides isolated PSM antigen nucleic acidmolecule operatively linked to a promoter of RNA transcription. Theisolated PSM antigen sequence can be linked to vector systems. Variousvectors including plasmid vectors, cosmid vectors, bacteriophage vectorsand other viruses are well known to ordinary skilled practitioners. Thisinvention further provides a vector which comprises the isolated nucleicacid molecule encoding for the PSM antigen.

As an example to obtain these vectors, insert and vector DNA can both beexposed to a restriction enzyme to create complementary ends on bothmolecules which base pair with each other and are then ligated togetherwith DNA ligase. Alternatively, linkers can be ligated to the insert DNAwhich correspond to a restriction site in the vector DNA, which is thendigested with the restriction enzyme which cuts at that site. Othermeans are also available and known to an ordinary skilled practitioner.

In an embodiment, the PSM sequence is cloned in the Not I/Sal I site ofpSPORT/vector (Gibco®—BRL). This plasmid, p55A-PSM, was deposited onAug. 14, 1992 with the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganism for the Purposes of Patent Procedure. Plasmid, p55A-PSM,was accorded ATCC Accession Number 75294.

This invention further provides a host vector system for the productionof a polypeptide having the biological activity of the prostate-specificmembrane antigen. These vectors may be transformed into a suitable hostcell to form a host cell vector system for the production of apolypeptide having the biological activity of PSM antigen.

Regulatory elements required for expression include is promotersequences to bind RNA polymerase and transcription initiation sequencesfor ribosome binding. For example, a bacterial expression vectorincludes a promoter such as the lac promoter and for transcriptioninitiation the Shine-Dalgarno sequence and the start codon AUG (14).Similarly, a eukaryotic expression vector includes a heterologous orhomologous promoter for RNA polymerase II, a downstream polyadenylationsignal, the start codon AUG, and a termination codon for detachment ofthe ribosome. Such vectors may be obtained commercially or assembledfrom the sequences described by methods well known in the art, forexample the methods described above for constructing vectors in general.Expression vectors are useful to produce cells that express the PSMantigen.

This invention further provides an isolated DNA or cDNA moleculedescribed hereinabove wherein the host cell is selected from the groupconsisting of bacterial cells (such as E. coli), yeast cells, fungalcells, insect cells and animal cells. Suitable animal cells include, butare not limited to Vero cells, HeLa cells, Cos cells, CV1 cells andvarious primary mammalian cells.

This invention further provides a method of producing a polypeptidehaving the biological activity of the prostate-specific membrane antigenwhich comprising growing host cells of a vector system containing thePSM antigen sequence under suitable conditions permitting production ofthe polypeptide and recovering the polypeptide so produced.

This invention provides a mammalian cell comprising a DNA moleculeencoding a mammalian PSM antigen, such as a mammalian cell comprising aplasmid adapted for expression in a mammalian cell, which comprises aDNA molecule encoding a mammalian PSM antigen and the regulatoryelements necessary for expression of the DNA in the mammalian cell solocated relative to the DNA encoding the mammalian PSM antigen as topermit expression thereof.

Numerous mammalian cells may be used as hosts, including, but notlimited to, the mouse fibroblast cell NIH3T3, CHO cells, HeLa cells, Ltkcells, Cos cells, etc. Expression plasmids such as that described supramay be used to transfect mammalian cells by methods well known in theart such as calcium phosphate precipitation, electroporation or DNAencoding the mammalian PSM antigen may be otherwise introduced intomammalian cells, e.g., by microinjection, to obtain mammalian cellswhich comprise DNA, e.g., cDNA or a plasmid, encoding a mammalian PSMantigen.

This invention provides a method for determining whether a ligand canbind to a mammalian prostate-specific membrane antigen which comprisescontacting a mammalian cell comprising an isolated DNA molecule encodinga mammalian prostate-specific membrane antigen with the ligand underconditions permitting binding of ligands to the mammalianprostate-specific membrane antigen, and thereby determining whether theligand binds to a mammalian prostate-specific membrane antigen.

This invention further provides ligands bound to the mammalian PSMantigen.

This invention also provides a therapeutic agent comprising a ligandidentified by the above-described method and a cytotoxic agentconjugated thereto. The cytotoxic agent may either be a radioisotope ora toxin. Examples of radioisotopes or toxins are well known to one ofordinary skill in the art.

This invention also provides a method of imaging prostate cancer inhuman patients which comprises administering to the patients at leastone ligand identified by the above-described method, capable of bindingto the cell surface of the prostate cancer cell and labelled with animaging agent under conditions permitting formation of a complex betweenthe ligand and the cell surface PSM antigen. This invention furtherprovides a composition comprising an effective imaging agent of the PSMantigen ligand and a pharmaceutically acceptable carrier.Pharmaceutically acceptable carriers are well known to one of ordinaryskill in the art. For an example, such a pharmaceutically acceptablecarrier can be physiological saline.

Also provided by this invention is a purified mammalian PSM antigen. Asused herein, the term “purified prostate-specific membrane antigen”shall mean isolated naturally-occurring prostate-specific membraneantigen or protein (purified from nature or manufactured such that theprimary, secondary and tertiary conformation, and posttranslationalmodifications are identical to naturally-occurring material) as well asnon-naturally occurring polypeptides having a primary structuralconformation (i.e. continuous sequence of amino acid residues). Suchpolypeptides include derivatives and analogs.

This invention further provides a polypeptide encoded by the isolatedmammalian nucleic acid sequence of PSM antigen.

It is believed that there may be natural ligand interacting with the PSMantigen. This invention provides a method to identify such naturalligand or other ligand which can bind to the PSM antigen. A method toidentify the ligand comprises a) coupling the purified mammalian PSMantigen to a solid matrix, b) incubating the coupled purified mammalianPSM protein with the potential ligands under the conditions permittingbinding of ligands and the purified PSM antigen; c) washing the ligandand coupled purified mammalian PSM antigen complex formed in b) toeliminate the nonspecific binding and impurities and finally d) elutingthe ligand from the bound purified mammalian PSM antigen. The techniquesof coupling proteins to a solid matrix are well known in the art.Potential ligands may either be deduced from the structure of mammalianPSM or by other empirical experiments known by ordinary skilledpractitioners. The conditions for binding may also easily be determinedand protocols for carrying such experimentation have long been welldocumented (15). The ligand-PSM antigen complex will be washed. Finally,the bound ligand will be eluted and characterized. Standard ligandscharacterization techniques are well known in the art.

The above method may also be used to purify ligands from any biologicalsource. For purification of natural ligands in the cell, cell lysates,serum or other biological samples will be used to incubate with themammalian PSM antigen bound on a matrix. Specific natural ligand willthen be identified and purified as above described.

With the protein sequence information, antigenic areas may be identifiedand antibodies directed against these areas may be generated andtargeted to the prostate cancer for imaging the cancer or therapies.

This invention provides an antibody directed against the amino acidsequence of a mammalian PSM antigen.

This invention provides a method to select specific regions on the PSMantigen to generate antibodies. The protein sequence may be determinedfrom the PSM DNA sequence. Amino acid sequences may be analyzed bymethods well known to those skilled in the art to determine whether theyproduce hydrophobic or hydrophilic regions in the proteins which theybuild. In the case of cell membrane proteins, hydrophobic regions arewell known to form the part of the protein that is inserted into thelipid bilayer of the cell membrane, while hydrophilic regions arelocated on the cell surface, in an aqueous environment. Usually, thehydrophilic regions will be more immunogenic than the hydrophobicregions. Therefore the hydrophilic amino acid sequences may be selectedand used to generate antibodies specific to mammalian PSM antigen. Foran example, hydrophilic sequences of the human PSM antigen shown inhydrophilicity plot of FIG. 16 may be easily selected. The selectedpeptides may be prepared using commercially available machines. As analternative, DNA, such as a cDNA or a fragment thereof, may be clonedand expressed and the resulting polypeptide recovered and used as animmunogen.

Polyclonal antibodies against these peptides may be produced byimmunizing animals using the selected peptides. Monoclonal antibodiesare prepared using hybridoma technology by fusing antibody producing Bcells from immunized animals with myeloma cells and selecting theresulting hybridoma cell line producing the desired antibody.Alternatively, monoclonal antibodies may be produced by in vitrotechniques known to a person of ordinary skill in the art. Theseantibodies are useful to detect the expression of mammalian PSM antigenin living animals, in humans, or in biological tissues or fluidsisolated from animals or humans.

In one embodiment, peptides Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID No. 35),Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No. 36) and Lys-Ser-Pro-Asp-Glu-Gly (SEQID No. 37) of human PSM antigen are selected.

This invention further provides polyclonal and monoclonal antibody(ies)against peptides Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID No. 35),Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No. 36) and Lys-Ser-Pro-Asp-Glu-Gly (SEQID No. 37).

This invention provides a therapeutic agent comprising antibodies orligand(s) directed against PSM antigen and a cytotoxic agent conjugatedthereto or antibodies linked enzymes which activate prodrug to kill thetumor. The cytotoxic agent may either be a radioisotope or toxin.

This invention provides a method of imaging prostate cancer in humanpatients which comprises administering to the patient the monoclonalantibody directed against the peptide of the mammalian PSM antigencapable of binding to the cell surface of the prostate cancer cell andlabeled with an imaging agent under conditions permitting formation of acomplex between the monoclonal antibody and the cell surfaceprostate-specific membrane antigen. The imaging agent is a radioisotopesuch as Indium¹¹¹.

This invention further provides a prostate cancer specific imaging agentcomprising the antibody directed against PSM antigen and a radioisotopeconjugated thereto.

This invention also provides a composition comprising an effectiveimaging amount of the antibody directed against the PSM antigen and apharmaceutically acceptable carrier. The methods to determine effectiveimaging amounts are well known to a skilled practitioner. One method isby titration using different amounts of the antibody.

This invention further provides an immunoassay for measuring the amountof the prostate-specific membrane antigen in a biological samplecomprising steps of a) contacting the biological sample with at leastone antibody directed against the PSM antigen to form a complex withsaid antibody and the prostate-specific membrane antigen, and b)measuring the amount of the prostate-specific membrane antigen in saidbiological sample by measuring the amount of said complex. One exampleof the biological sample is a serum sample.

This invention provides a method to purify mammalian prostate-specificmembrane antigen comprising steps of a) coupling the antibody directedagainst the PSM antigen to a solid matrix; b) incubating the coupledantibody of a) with lysate containing prostate-specific membrane antigenunder the condition which the antibody and prostate membrane specificcan bind; c) washing the solid matrix to eliminate impurities and d)eluting the prostate-specific membrane antigen from the coupledantibody.

This invention also provides a transgenic nonhuman mammal whichcomprises the isolated nucleic acid molecule encoding a mammalian PSMantigen. This invention further provides a transgenic nonhuman mammalwhose genome comprises antisense DNA complementary to DNA encoding amammalian prostate-specific membrane antigen so placed as to betranscribed into antisense mRNA complementary to mRNA encoding theprostate-specific membrane antigen and which hybridizes to mRNA encodingthe prostate specific antigen thereby reducing its translation.

Animal model systems which elucidate the physiological and behavioralroles of mammalian PSM antigen are produced by creating transgenicanimals in which the expression of the PSM antigen is either increasedor decreased, or the amino acid sequence of the expressed PSM antigen isaltered, by a variety of techniques. Examples of these techniquesinclude, but are not limited to: 1) Insertion of normal or mutantversions of DNA encoding a mammalian PSM antigen, by microinjection,electroporation, retroviral transfection or other means well known tothose skilled in the art, into appropriate fertilized embryos in orderto produce a transgenic animal (16) or 2) Homologous recombination (17)of mutant or normal, human or animal versions of these genes with thenative gene locus in transgenic animals to alter the regulation ofexpression or the structure of these PSM antigen sequences. Thetechnique of homologous recombination is well known in the art. Itreplaces the native gene with the inserted gene and so is useful forproducing an animal that cannot express native PSM antigen but doesexpress, for example, an inserted mutant PSM antigen, which has replacedthe native PSM antigen in the animal's genome by recombination,resulting in underexpression of the transporter. Microinjection addsgenes to the genome, but does not remove them, and so is useful forproducing an animal which expresses its own and added PSM antigens,resulting in overexpression of the PSM antigens.

One means available for producing a transgenic animal, with a mouse asan example, is as follows: Female mice are mated, and the resultingfertilized eggs are dissected out of their oviducts. The eggs are storedin an appropriate medium such as M2 medium (16). DNA or cDNA encoding amammalian PSM antigen is purified from a vector by methods well known inthe art. Inducible promoters may be fused with the coding region of theDNA to provide an experimental means to regulate expression of thetrans-gene. Alternatively or in addition, tissue specific regulatoryelements may be fused with the coding region to permit tissue-specificexpression of the trans-gene. The DNA, in an appropriately bufferedsolution, is put into a microinjection needle (which may be made fromcapillary tubing using a pipet puller) and the egg to be injected is putin a depression slide. The needle is inserted into the pronucleus of theegg, and the DNA solution is injected. The injected egg is thentransferred into the oviduct of a pseudopregnant mouse (a mousestimulated by the appropriate hormones to maintain pregnancy but whichis not actually pregnant), where it proceeds to the uterus, implants,and develops to term. As noted above, microinjection is not the onlymethod for inserting DNA into the egg cell, and is used here only forexemplary purposes.

Another use of the PSM antigen sequence is to isolate homologous gene orgenes in different mammals. The gene or genes can be isolated by lowstringency screening of either cDNA or genomic libraries of differentmammals using probes from PSM sequence. The positive clones identifiedwill be further analyzed by DNA sequencing techniques which are wellknown to an ordinary person skilled in the art. For example, thedetection of members of the protein serine kinase family by homologyprobing (18).

This invention provides a method of suppressing or modulating metastaticability of prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells comprising introducing a DNA molecule encoding aprostate specific membrane antigen operatively linked to a 5′ regulatoryelement into a tumor cell of a subject, in a way that expression of theprostate specific membrane antigen is under the control of theregulatory element, thereby suppressing or modulating metastatic abilityof prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells. The subject may be a mammal or more specifically ahuman.

In one embodiment, the DNA molecule encoding prostate specific membraneantigen operatively linked to a 5′ regulatory element forms part of atransfer vector which is inserted into a cell or organism. In additionthe vector is capable or replication and expression of prostate specificmembrane antigen. The DNA molecule encoding prostate specific membraneantigen can be integrated into a genome of a eukaryotic or prokaryoticcell or in a host cell containing and/or expressing a prostate specificmembrane antigen.

Further, the DNA molecule encoding prostate specific membrane antigenmay be introduced by a bacterial, viral, fungal, animal, or liposomaldelivery vehicle. Other means are also available and known to anordinary skilled practitioner.

Further, the DNA molecule encoding a prostate specific membrane antigenoperatively linked to a promoter or enhancer. A number of viral vectorshave been described including those made from various promoters andother regulatory elements derived from virus sources. Promoters consistof short arrays of nucleic acid sequences that interact specificallywith cellular proteins involved in transcription. The combination ofdifferent recognition sequences and the cellular concentration of thecognate transcription factors determines the efficiency with which agene is transcribed in a particular cell type.

Examples of suitable promoters include a viral promoter. Viral promotersinclude: adenovirus promoter, an simian virus 40 (SV40) promoter, acytomegalovirus CCMV) promoter, a mouse mammary tumor virus (MMTV)promoter, a Malony murine leukemia virus promoter, a murine sarcomavirus promoter, and a Rous sarcoma virus promoter.

Further, another suitable promoter is a heat shock promoter.Additionally, a suitable promoter is a bacteriophage promoter. Examplesof suitable bacteriophage promoters include but not limited to, a T7promoter, a T3 promoter, an SP6 promoter, a lambda promoter, abaculovirus promoter.

Also suitable as a promoter is an animal cell promoter such as aninterferon promoter, a metallothionein promoter, an immunoglobulinpromoter. A fungal promoter is also a suitable promoter. Examples offungal promoters include but are not limited to, an ADC1 promoter, anARG promoter, an ADH promoter, a CYC1 promoter, a CUP promoter, an ENO1promoter, a GAL promoter, a PRO promoter, a PGK promoter, a GAPDHpromoter, a mating type factor promoter. Further, plant cell promotersand insect cell promoters are also suitable for the methods describedherein.

This invention provides a method of suppressing or modulating metastaticability of prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells, comprising introducing a DNA molecule encoding aprostate specific membrane antigen operatively linked to a 5′ regulatoryelement coupled with a therapeutic DNA into a tumor cell of a subject,thereby suppressing or modulating metastatic ability of prostate tumorcells, prostate tumor growth or elimination of prostate tumor cells. Thesubject may be a mammal or more specifically a human.

Further, the therapeutic DNA which is coupled to the DNA moleculeencoding a prostate specific membrane antigen operatively linked to a 5′regulatory element into a tumor cell may code for a cytokine, viralantigen, or a pro-drug activating enzyme. Other means are also availableand known to an ordinary skilled practitioner.

The cytokine used may be interleukin-2, interleukin-12, interferonalpha, beta or gamma, granulocytic macrophage—colony stimulating factor,or other immunity factors.

In addition, this invention provides a prostate tumor cell, comprising aDNA molecule isolated from mammalian nucleic acid encoding a mammalianprostate-specific membrane antigen under the control of a prostatespecific membrane antigen operatively linked to a 5′ regulatory element.

As used herein, DNA molecules include complementary DNA (cDNA),synthetic DNA, and genomic DNA.

This invention provides a therapeutic vaccine for preventing humanprostate tumor growth or stimulation of prostate tumor cells in asubject, comprising administering an effective amount to the prostatecell, and a pharmaceutical acceptable carrier, thereby preventing thetumor growth or stimulation of tumor cells in the subject. Other meansare also available and known to an ordinary skilled practitioner.

This invention provides a method of detecting hematogenous micrometastictumor cells of a subject, comprising (A) performing nested polymerasechain reaction (PCR) on blood, bone marrow or lymph node samples of thesubject using the prostate specific membrane antigen primers, and (B)verifying micrometastases by DNA sequencing and Southern analysis,thereby detecting hematogenous micrometastic tumor cells of the subject.The subject may be a mammal or more specifically a human.

The micrometastatic tumor cell may be a prostatic cancer and the DNAprimers may be derived from prostate specific antigen. Further, thesubject may be administered with simultaneously an effective amount ofhormones, so as to increase expression of prostate specific membraneantigen.

This invention provides a method of abrogating the mitogenic responsedue to transferrin, comprising introducing a DNA molecule encodingprostate specific membrane antigen operatively linked to a 5′ regulatoryelement into a tumor cell, the expression of which gene is directlyassociated with a defined pathological effect within a multicellularorganism, thereby abrogating mitogen response due to transferrin. Thetumor cell may be a prostate cell.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS First Series of Experiments

Materials and Methods

The approach for cloning the gene involved purification of the antigenin large quantities by immunoprecipitation, and microsequencing ofseveral internal peptides for use in synthesizing degenerateoligonucleotide primers for subsequent use in the polymerase chainreaction (19, 20). A partial cDNA was amplified as a PCR product andthis was used as a homologous probe to clone the full-length cDNAmolecule from a LNCaP (Lymph Node Carcinoma of Prostate) cell line cDNAplasmid library (8). Early experiments revealed to us that the CYT-356antibody (9) was not capable of detecting the antigen produced inbacteria since the epitope was the glycosylated portion of the PSMantigen, and this necessitated our more difficult, yet elaborateapproach.

Western Analysis of the PSM Antigen

Membrane proteins were isolated from cells by hypotonic lysis followedby centrifugation over a sucrose density gradient (21). 10-20 μg ofLNCaP, DU-145, and PC-3 membrane proteins were electrophoresed through a10% SDS-PAGE resolving gel with a 4% stacking gel at 9-10 milliamps for16-18 hours. Proteins were electroblotted onto PVDF membranes(Millipore® Corp.) in transfer buffer (48 mM Tris base, 39 mM Glycine,20% Methanol) at 25 volts overnight at 4° C. Membranes were blocked inTSB (0.15M NaCl, 0.01M Tris base, 5% BSA) for 30 minutes at roomtemperature followed by incubation with 10-15 μg/ml of CYT-356monoclonal antibody (Cytogen Corp.) for 2 hours. Membranes were thenincubated with 10-15 μg/ml of rabbit anti-mouse immunoglobulin (AccurateScientific) for 1 hour at room temperature followed by incubation with¹²⁵I-Protein A (Amersham®) at 1×10⁶ cpm/ml at room temperature.Membranes were then washed and autoradiographed for 12-24 hours at −70°C. (FIG. 1).

Immunohistochemical Analysis of PSM Antigen Expression

The avidin-biotin method of immunohistochemical detection was employedto analyze both human tissue sections and cell lines for PSM Antigenexpression (22). Cryostat-cut prostate tissue sections (4-6% thick) werefixed in methanol/acetone for 10 minutes. Cell cytospins were made onglass slides using 50,000 cells/100 μl/slide. Samples were treated with1% hydrogen peroxide in PBS for 10-15 minutes in order to remove anyendogenous peroxidase activity. Tissue sections were washed severaltimes in PBS, and then incubated with the appropriate suppressor serumfor 20 minutes. The suppressor serum was drained off and the sections orcells were then incubated with the diluted CYT-356 monoclonal antibodyfor 1 hour. Samples were then washed with PBS and sequentially incubatedwith secondary antibodies (horse or goat immunoglobulins, 1:200 dilutionfor 30 minutes), and with avidin-biotin complexes (1:25 dilution for 30minutes). DAB was used as a chromogen, followed by hematoxylincounterstaining and mounting. Frozen sections of prostate samples andduplicate cell cytospins were used as controls for each experiment. As apositive control, the anti-cytokeratin monoclonal antibody CAM 5.2 wasused following the same procedure described above. Tissue sections areconsidered by us to express the PSM antigen if at least 5% of the cellsdemonstrate immunoreactivity. Our scoring system is as follows: 1=<5%;2=5-19%; 3=20-75%; and 4=>75% positive cells. Homogeneity versusheterogeneity was accounted for by evaluating positive and negativecells in 3-5 high power light microscopic fields (400×, recording thepercentage of positive cells among 100-500 cells. The intensity ofimmunostaining is graded on a 1+ to 4+ scale, where 1-represents mild,2-3+ represents moderate, and 4+ represents intense immunostaining ascompared to positive controls.

Immunoprecipitation of the PSM Antigen

80%-confluent LNCaP cells in 100 mm petri dishes were starved in RPMImedia without methionine for 2 hours, after which ³³S-Methionine wasadded at 100 μCi/ml and the cells were grown for another 16-18 hours.Cells were then washed and lysed by the addition of 1 ml of lysis buffer(1% Triton X-100, 50 mM Hepes pH 7.5, 10% glycerol, 150 MM MgCl₂, 1 mMPMSF, and 1 mM EGTA) with incubation for 20 minutes at 40° C. Lysateswere pre-cleared by mixing with Pansorbin® cells (Calbiochem) for 90minutes at 4° C. Cell lysates were then mixed with Protein A Sepharose®CL-4B beads (Pharmacia®) previously bound with CYT-356 antibody (CytogenCorp.) and RAM antibody (Accurate Scientific) for 3-4 hours at 4° C. 12μg of antibody was used per 3 mg of beads per petri dish. Beads werethen washed with HNTG buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 0.1%Triton X-100, 10% glycerol, and 2 mM Sodium Orthovanadate), resuspendedin sample loading buffer containing mercaptoethanol, denatured at 95° C.for 5-10 minutes and run on a 10% SDS-PAGE gel with a 4° stacking gel at10 milliamps overnight. Gels were stained with Coomassie Blue, destainedwith acetic acid/methanol, and dried down in a vacuum dryer at 60° C.Gels were then autoradiographed for 16-24 hours at −70° C. (FIG. 2 A-D).

Large-Scale Immunoprecigitation and Peptide Sequencing

The procedure described above for immunoprecipitation was repeated with8 confluent petri dishes containing approximately 6×10⁷ LNCaP cells. Theimmunoprecipitation product was pooled and loaded into two lanes of a10% SDS-PAGE gel and electrophoresed at 9-10 milliamps for 16 hours.Proteins were electroblotted onto Nitrocellulose BA-85 membranes(Schleicher and Schuell®) for 2 hours at 75 volts at 4° C. in transferbuffer. Membranes were stained with Ponceau Red to visualize theproteins and the 100 KD protein band was excised, solubilized, anddigested proteolytically with trypsin. HPLC was then performed on thedigested sample on an Applied Biosystems Model 171C and clear dominantpeptide peaks were selected and sequenced by modified Edman degradationon a modified post liquid Applied Biosystems Model 477A Protein/PeptideMicrosequencer (23). Sequencing data on all of the peptides is includedwithin this document. We attempted to sequence the amino-terminus of thePSM antigen by a similar method which involved purifying the antigen byimmunoprecipitation and transfer via electro-blotting to a PVDF membrane(Millipore®). Protein was analyzed on an Applied Biosystems Model 477AProtein/Peptide Sequencer and the amino terminus was found to beblocked, and therefore no sequence data could be obtained by thistechnique.

PSM Antigen Peptide Sequences: 2T17 SLYES(W)TK (SEQ ID No. 3) #5 2T22(S)YPDGXNLPGG(g)VQR (SEQ ID No. 4) #9 2T26 FYDPMFK (SEQ ID No. 5) #32T27 IYNVIGTL(K) (SEQ ID No. 6) #4 2T34 FLYXXTQIPHLAGTEQNFQLAK (SEQ IDNO. 7) #6 2T35 G/PVILYSDPADYFAPD/GVK (SEQ ID No. 8, 9) #2 2T38AFIDPLGLPDRPFYR (SEQ ID No. 10) #1 2146 YAGESFPGIYDALFDIESK (SEQ ID No.11) #8 2T47 TILFAS(W)DAEEFGXX(q)STE(e)A(E) . . . (SEQ ID No. 12) #7Notes: X means that no residue could be identified at this position.Capital denotes identification but with a lower degree of confidence.(lower case) means residue present but at very low levels. . . .indicates sequence continues but has dropped below detection limit.

All of these peptide sequences were verified to be unique after acomplete homology search of the translated Genbank computer database.

Degenerate PCR

Sense and anti-sense 5′-unphosphorylated degenerate oligonucleotideprimers 17 to 20 nucleotides in length corresponding to portions of theabove peptides were synthesized on an Applied Biosystems Model 394A DNASynthesizer. These primers have degeneracies from 32 to 144. The primersused are shown below. The underlined amino acids in the peptidesrepresent the residues used in primer design.

Peptide 3: (SEQ ID No. 5)

PSM Primer “A” TT(C or T)—TA(C or T)—GA(C or T)—CCX—ATG—TT (SEQ IDNo.13)

PSM Primer “B” AAC—ATX—GG(A or G)—TC(A or G)—TA(A or G)—AA (SEQ ID No.14)

Primer A is sense primer and B is anti-sense. Degeneracy is 32-fold.

Peptide 4: IYNVIGTL(K) (SEQ ID No. 6)

PSM Primer “C” AT(T or C or A)—TA(T or C)—AA(T or C)—GTX—AT(T or C orA)—GG (SEQ ID No. 15)

PSM Primer “D” CC(A or T or G)—ATX&13 AC(G or A)—TT(A or G)—TA(A or G orT)—AT (SEQ ID No. 16)

Primer C is sense primer and D is anti-sense. Degeneracy is 144-fold.

Peptide 2: G/PVILYSDPADYFAPD/GVK (SEQ ID No. 8,9)

PSM Primer “E” CCX—GCX—GA(T or C)—TA(T or C)—TT(T or C)—CC (SEQ ID No.17)

PSM Primer “F” GC(G or A)—AA(A or G)—TA(A or G)—TXC—GCX—GG (SEQ ID No.16)

Primer E is sense primer and F is antisense primer. Degeneracy is128-fold.

Peptide 6. FLYXXTQIPHLAGTEONFQLAK (SEQ ID No. 7)

PSM Primer “I” ACX—GA(A or G)—CA(A or G)—AA(T or C)—TT(T or C)—CA(A orG)—CT (SEQ ID No. 19)

PSM Primer “J” AG—(T or C)TG—(A or G)AA—(A or G)TT—(T or C)TG—(T orC)TC—XGT (SEQ ID No. 20)

PSM Primer “K” GA(A or G)—CA(A or G)—AA(T or C)—TT(T or C) CA(A or G)—CT(SEQ ID No. 21)

PSM Primer “L” AG—(T or C)TG—(A or G)AA—(A or G)TT—(T or C)TG—(T or C)TC(SEQ ID No. 22)

Primers I and K are sense printers and J and L are anti-sense. I and Jhave degeneracies of 128-fold and K and L have 32-fold degeneracy.

Peptide 7: TILFAS(W)DAEEPGXX(q)STE(e)A(E) . . . (SEQ ID No. 12)

PSM Primer “M” TGG—GA(T or C)—GCX—GA(A or G)—GA(A or G)—TT(C or T)—GG(SEQ ID No. 23)

PSM Primer “N” CC—(G or A)AA—(T or C)TC—(T or C)TC—XGC—(A or G)TC—CCA(SEQ ID No. 24)

PSM Primer row TGG—GA(T or C)—GCX—GA(A or G)—GA(A or G)—TT (SEQ ID No.25)

PSM Primer “p” AA—(T or C)TC—(T or C)TC—XGC—(A or G)TC—CCA (SEQ ID No.26)

Primers M and O are sense primers and N and P are anti-sense. M and Nhave degeneracy of 64-fold and O and P are 32- fold degenerate.

Degenerate PCR was performed using a Perkin-Elmer Model 480 DNA thermalcycler. cDNA template for the PCR was prepared from LNCaP mRNA which hadbeen isolated by standard methods of oligo dT chromatography(Collaborative Research). The cDNA synthesis was carried out as follows:

4.5 μl LNCaP poly A+RNA (2 μg)

1.0 μl Oligo dT primers (0.5 μg)

4.5 μl dH₂O

10 μl

Incubate at 68° C.×10 minutes.

Quick chill on ice ×5 minutes.

Add:

4 μl 5 × RT Buffer

2 μl 0.1M DTT

1 μl 10 mM dNTPs

0.5 μl RNasin (Promega)

1.5 μl dH₂O

19 μl

Incubate for 2 minutes at 37° C.

Add 1 μl Superscript® Reverse Transcriptase (Gibco®-BRL) Incubate for 1hour at 37° C.

Add 30 μl dH₂.

Use 2 μl per PCR reaction.

Degenerate PCR reactions were optimized by varying the annealingtemperatures, Mg++ concentrations, primer concentrations, buffercomposition, extension times and number of cycles. Our optimal thermalcycler profile was: Denaturation at 94° C.×30 seconds, Annealing at45-55° C. for 1 minute (depending on the mean T_(m) of the primersused), and Extension at 72° C. for 2 minutes.

5 μl 10 × PCR Buffer*

5 μl 2.5 mM dNTP Mix

5 μl Primer Mix (containing 0.5-1.0 g each of sense d anti-senseprimers)

5 μl 100 mM β-mercaptoethanol

2 μl LNCaP cDNA template

5 μl 25 mM MgCl₂ (2.5 mM final)

21 μl dH₂O

20 μl diluted Tag Polymerase (0.5U/μl)

50 μl total volume

Tubes were overlaid with 60 μl of light mineral oil and amplified for 30cycles. PCR products were analyzed by electrophoresing 5 μl of eachsample on a 2-3% agarose gel followed by staining with Ethidium bromideand photography.

*10× PCR Buffer

166 mM NH₄SO₄

670 mM Tris, pH 8.8

2 mg/ml BSA

Representative photographs displaying PCR products are shown in FIG. 5.

Cloning of PCR Products

In order to further analyze these PCR products, these products-werecloned into a suitable plasmid vector using “TA Cloning” (Invitrogen®Corp.). The cloning strategy employed here is to directly ligate PCRproducts into a plasmid vector possessing overhanging T residues at theinsertion site, exploiting the fact that Tag polymerase leavesoverhanging A residues at the ends of the PCR products. The ligationmixes are transformed into competent E. coli cells and resultingcolonies are grown up, plasmid DNA is isolated by the alkaline lysismethod (24), and screened by restriction analysis (FIG. 6 A-B).

DNA Sequencing of PCR Products

TA Clones of PCR products were then sequenced by the dideoxy method (25)using Sequenase (U.S. Biochemical). 3-4 μg of each plasmid DNA wasdenatured with NaOH and ethanol precipitated. Labeling reactions werecarried out as per the manufacturers recommendations using ³⁵S-ATP, andthe reactions were terminated as per the same protocol. Sequencingproducts were then analyzed on 6% polyacrylamide/7M Urea gels using anIBI sequencing apparatus. Gels were run at 120 watts for 2 hours.Following electrophoresis, the gels were fixed for 15-20 minutes in 10%methanol/10% acetic acid, transferred onto Whatman 3MM paper and drieddown in a Biorad® vacuum dryer at 80° C. for 2 hours. Gels were thenautoradiographed at room temperature for 16-24 hours. In order todetermine whether the PCR products were the correct clones, we analyzedthe sequences obtained at the 5′ and 3′ ends of the molecules lookingfor the correct primer sequences, as well as adjacent sequences whichcorresponded to portions of the peptides not used in the design of theprimers.

IN-20 was confirmed to be correct and represent a partial cDNA for thePSM gene. In this PCR reaction, I and N primers were used. The DNAsequence we obtained when reading from the I primer was:

(SEQ ID No. 30) ACG GAG CAA AJLC TTT CAG CTT GCA AAG (SEQ ID No. 31)T E O N P O L A X

The underlined amino acids were the portion of peptide 6 that was usedto design this sense primer and the remaining amino acids which agreewith those present within our peptide confirm that this end of themolecule represents the correct protein (PSM antigen).

When we analyzed the other end of the molecule by reading from the Nprimer the sequence was:

(SEQ ID No. 32) CTC TTC GGC ATC CCA GGT TGC ALAA CAA ATT TGT TCT

Since this represents the anti-sense DNA sequence, we need to show thecomplementary sense sequence in order to find our peptide.

Sense Sequence:

(SEQ ID No. 33) AGA ACA ATT TTG TTT GCK AGC TGG GAT GCC AAG GAG(SEQ ID No. 34) R T I L P A S W D A E B

The underlined amino acids here represent the portion of peptide 7 usedto create primer N. All of the amino acids upstream of this primer arecorrect in the IN-20 clone, agreeing with the amino acids found inpeptide 7. Further DNA sequencing has enabled us to identify thepresence of our other PSM peptides within the DNA sequence of ourpositive clone.

The DNA sequence of this partial cDKA was found to be unique whenscreened on the Genbank computer database.

cDNA Library Construction and Cloning of Full—Length PSM cDNA

A cDNA library from LNCaP mRNA was constructed using the Superscript®plasmid system (BRL®-Gibco). The library was transformed using competentDH5-α cells and plated onto 100 mm plates containing LB plus 100 μg/mlof Carbenicillin. Plates were grown overnight at 37° C. and colonieswere transferred to nitrocellulose filters. Filters were processedand-screened as per Grunstein and Hogness (26), using our 1.1 kb partialcDNA homologous probe which was radiolabelled with ³²P-dCTP by randompriming (27). We obtained eight positive colonies which upon DNArestriction and sequencing analysis proved to represent full-length cDNAmolecules coding for the PSM antigen. Shown in FIG. 7 is anautoradiogram showing the size of the cDNA molecules represented in ourlibrary and in FIG. 8 restriction analysis of several full-length clonesis shown. FIG. 9 is a plasmid Southern analysis of the samples in FIG.6, showing that they all hybridize to the 1.1 kb partial cDNA probe.

Both the cDNA as well as the antigen have been screened through theGenbank Computer database (Human Genome Project) and have been found tobe unique.

Northern Analysis of PSM Gene Expression

Northern analysis (28) of the PSM gene has revealed that expression islimited to the prostate and to prostate carcinoma.

RNA samples (either 10 μg of total RNA or 2 μg of poly A+RNA) weredenatured and electrophoresed through 1.1% agarose/formaldehyde gels at60 milliamps for 6-8 hours. RNA was then transferred to Nytran® nylonmembranes (Schleicher and Schuell®) by pressure blotting in 10× SSC witha Posi-blotter (Stratagene®). RNA was cross-linked to the membranesusing a Stratalinker (Stratagene®) and subsequently baked in a vacuumoven at 80° C. for 2 hours. Blots were pre-hybridized at 65° C. for 2hours in prehybridization solution (BRL®) and subsequently hybridizedfor 16 hours in hybridization buffer (BRL®) containing 1-2×10⁶ cpm/ml of³²P-labelled random-primed cDNA probe. Membranes were washed twice in 1×SSPE/1% SDS and twice in 0.1 × SSPE/1% SDS at 42° C. Membranes were thenair-dried and autoradiographed for 12-36 hours at −70° C.

PCR Analysis of PSM Gene Expression in Human Prostate Tissues

PCR was performed on 15 human prostate samples to determine PSM geneexpression. Five samples each from normal prostate tissue, benignprostatic hyperplasia, and prostate cancer were used (histologyconfirmed by MSKCC Pathology Department).

10 μg of total RNA from each sample was reverse transcribed to made cDNAtemplate as previously described in section IV. The primers usedcorresponded to the 5 and 3′ ends of our 1.1 kb partial cDRA, IN-20, andtherefore the expected size of the amplified band is 1.1 kb. Since theT_(m) of our primers is 64° C. we annealed the primers in our PCR at 60°C. We carried out the PCR for 35 cycles using the same conditionspreviously described in section IV.

LNCaP and H26—Ras transfected LNCaP (29) were included as a positivecontrol and DU-145 as a negative control. 14/15 samples clearlyamplified the 1.1 kb band and therefore express the gene.

Experimental Results

The gene which encodes the 100 kD PSM antigen has been identified. Thecomplete cDNA sequence is shown in Sequence ID #1. Underneath thatnucleic acid sequence is the predicted translated amino acid sequence.The total number of the amino acids is 750, ID.#2. The hydrophilicity ofthe predicted protein sequence is shown in FIG. 16. Shown in FIG. 17 arethree peptides with the highest point of hydrophilicity. They are:Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID No. 35); Asn-Glu-Asp-Gly-Asn-Glu (SEQ IDNo. 36; and Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID No. 37).

By the method of Klein, Kanehisa and DeLisi, a specificmembrane-spanning domain is identified. The sequence is from the aminoacid #19 to amino acid #44:Ala-Gly-Ala-Leu-Val-Leu-Aal-Gly-Gly-Phe-Phe-Leu-Leu-Gly-Phe-Leu-Phe (SEQID No. 38).

This predicted membrane-spanning domain was computed on PC Gene(computer software program). This data enables prediction of inner andouter membrane domains of the PSM antigen which aids in designingantibodies for uses in targeting and imaging prostate cancer.

When the PSM antigen sequence with other known sequences of the GeneBankwere compared, homology between the PSM antigen sequence and thetransferrin receptor sequence were found. The data are shown in FIG. 18.

EXPERIMENTAL DISCUSSIONS

Potential Uses for PSM Antigen:

-   -   1. Tumor detection:        Microscopic:

Unambiguous tumor designation can be accomplished by use of probes fordifferent antigens. For prostatic cancer, the PSM antigen probe mayprove beneficial. Thus PSM could be used for diagnostic purposes andthis could be accomplished at the microscopic level using in-situhybridization using sense (control) and antisense probes derived fromthe coding region of the cDNA cloned by the applicants. This could beused in assessment of local extraprostatic extension, involvement oflymph node, bone or other metastatic sites. As bone metastasis presentsa major problem in prostatic cancer, early detection of metastaticspread is required especially for staging. In some tumors detection oftumor cells in bone marrow portends a grim prognosis and suggests thatinterventions aimed at metastasis be tried. Detection of PSM antigenexpression in bone marrow aspirates or sections may provide such earlyinformation. PCR amplification or in-situ hybridization may be used.This could be developed for any possible metastatic region.

-   -   2. Antigenic site identification

The knowledge of the cDNA for the antigen also provides for theidentification of areas that would serve as good antigens for thedevelopment of antibodies for use against specific amino acid sequencesof the antigen. Such sequences may be at different regions such asoutside, membrane or inside of the PSM antigen. The development of thesespecific antibodies would provide for immunohistochemical identificationof the antigen. These derived antibodies could then be developed foruse, especially ones that work in paraffin fixed sections as well asfrozen section as they have the greatest utility for immunodiagnosis.

-   -   3. Restriction fragment length polymorphism and genomic DNA

Restriction fragment length polymorphisms (RFLPS) have proven to beuseful in documenting the progression of genetic damage that occursduring tumor initiation and promotion. It may be that RFLP analysis willdemonstrate that changes in PSM sequence restriction mapping may provideevidence of predisposition to risk or malignant potential or progressionof the prostatic tumor.

Depending on the chromosomal location of the PSM antigen, the PSMantigen gene may serve as a useful chromosome location marker forchromosome analysis.

-   -   4. Serum

With the development of antigen specific antibodies, if the antigen orselected antigen fragments appear in the serum they may provide for aserum marker for the presence of metastatic disease and be usefulindividually or in combination with other prostate specific markers.

-   -   5. Imaging

As the cDNA sequence implies that the antigen has the characteristics ofa membrane spanning protein with the majority of the protein on theexofacial surface, antibodies, especially monoclonal antibodies to thepeptide fragments exposed and specific to the tumor may provide fortumor imaging local extension of metastatic tumor or residual tumorfollowing prostatectomy or irradiation. The knowledge of the codingregion permits the generation of monoclonal antibodies and these can beused in combination to provide for maximal imaging purposes. Because theantigen shares a similarity with the transferrin receptor based on cDNAanalysis (approximately 54%), it may be that there is a specific normalligand for this antigen and that identification of the ligand(s) wouldprovide another means of imaging.

-   -   6. Isolation of ligands

The PSM antigen can be used to isolate the normal ligand(s) that bind toit. These ligand(s) depending on specificity may be used for targeting,or their serum levels may be predictive of disease status. If it isfound that the normal ligand for PSM is a carrier molecule then it maybe that PSM could be used to bind to that ligand for therapy purposes(like an iron chelating substance) to help remove the ligand from thecirculation. If the ligand promotes tumor growth or metastasis thenproviding soluble PSM antigen would remove the ligand from binding theprostate. Knowledge of PSM antigen structure could lend to generation ofsmall fragment that binds ligand which could serve the same purpose.

-   -   7. Therapeutic uses

a) Ligands. The knowledge that the cDNA structure of PSM antigen sharesstructural homology with the transferrin receptor (54% on the nucleicacid level) implies that there may be an endogenous ligand for thereceptor that may or may not be transferrin-like. Transferrin is thoughtto be a ligand that transports iron into the cell after binding to thetransferrin receptor. However, apotransferrin is being reported to be agrowth factor for some cells which express the transferrin receptor(30). Whether transferrin is a ligand for this antigen or some otherligand binds to this ligand remains to be determined. If a ligand isidentified it may carry a specific substance such as a metal ion (ironor zinc or other) into the tumor and thus serve as a means to delivertoxic substances (radioactive or cytotoxic chemical i.e. toxin likericin or cytotoxic alkylating agent or cytotoxic prodrug) to the tumor.

The main metastatic site for prostatic tumor is the bone. The bone andbone stroma are rich in transferrin. Recent studies suggest that thismicroenvironment is what provides the right “soil” for prostaticmetastasis in the bone (31). It may be that this also promotesattachment as well, these factors which reduce this ability may diminishprostatic metastasis to the bone and prostatic metastatic growth in thebone.

It was found that the ligand for the new antigen (thought to be anoncogene and marker of malignant phenotype in breast carcinoma) servedto induce differentiation of breast cancer cells and thus could serve asa treatment for rather than promotor of the disease. It may be thatligand binding to the right region of PSM whether with natural ligand orwith an antibody may serve a similar function.

Antibodies against PSM antigen coupled with a cytotoxic agent will beuseful to eliminate prostate cancer cells. Transferrin receptorantibodies with toxin conjugates are cytotoxic to a number of tumorcells as tumor cells tend to express increased levels of transferrinreceptor (32). Transferrin receptors take up molecules into the cell byendocytosis. Antibody drug combinations can be toxic. Transferrin linkedtoxin can be toxic.

b) Antibodies against PSM antigen coupled with a cytotoxic agent will beuseful to eliminate prostate cancer cells. The cytotoxic agent may be aradioisotope or toxin as known in ordinary skill of the art. The linkageof the antibody and the toxin or radioisotope can be chemical. Examplesof direct linked toxins are doxorubicin, chlorambucil, ricin,pseudomonas exotoxin etc., or a hybrid toxin can be generated ½ withspecificity for PSM and the other ½ with specificity for the toxin. Sucha bivalent molecule can serve to bind to the tumor and the other ½ todeliver a cytotoxic to the tumor or to bind to and activate a cytotoxiclymphocyte such as binding to the T₁-T₃ receptor complex. Antibodies ofrequired specificity can also be cloned into T cells and by replacingthe immunoglobulin domain of the T cell receptor (TcR); cloning in thedesired MAb heavy and light chains; splicing the U_(b) and U_(L) genesegments with the constant regions of the α and β TCR chains andtransfecting these chimeric Ab/TcR genes in the patients' T cells,propagating these hybrid cells and infusing them into the patient (33).Specific knowledge of tissue specific antigens for targets andgeneration of MAb's specific for such targets will help make this ausable approach. Because the PSM antigen coding region providesknowledge of the entire coding region, it is possible to generate anumber of antibodies which could then be used in combination to achievean additive or synergistic anti-tumor action. The antibodies can belinked to enzymes which can activate non-toxic prodrugs at its site ofthe tumor such as Ab-carboxypeptidase and 4-(bis(2 chloroethyl)amino)benzoyl-α-glutamic acid and its active parent drug in mice (34).

It is possible to produce a toxic genetic chimera such as TP-40 agenetic recombinant that possesses the cDNA from TGF-alpha and the toxicportion of pseudomonas exotoxin so the TGF and portion of the hybridbinds the epidermal growth factor receptor (EGFR), and the pseudomonasportion gets taken up into the cell enzymatically and inactivates theribosomes ability to perform protein synthesis resulting in cell death.When we know the ligand for the PSM antigen we can do the same.

In addition, once the ligand for the PSM antigen is identified, toxincan be chemically conjugated to the ligands. Such conjugated ligands canbe therapeutically useful. Examples of the toxins are daunomycin,chlorambucil, ricin, pseudomonas exotoxin, etc. Alternatively, chimericconstruct can be created linking the cDNA of the ligand with the cDNA ofthe toxin. An example of such toxin is TGFα and pseudomonas exotoxin(35).

-   -   8. Others

The PSM antigen may have other uses. It is well known that the prostateis rich in zinc, if the antigen provides function relative to this orother biologic function the PSM antigen may provide for utility in thetreatment of other prostatic pathologies such as benign hyperplasticgrowth and/or prostatitis.

Because purified PSM antigen can be generated, the purified PSM antigencan be linked to beads and use it like a standard “affinity”purification. Serum, urine or other biological samples can be used toincubate with the PSM antigen bound onto beads. The beads may be washedthoroughly and then eluted with salt or pH gradient. The eluted materialis SDS gel purified and used as a sample for microsequencing. Thesequences will be compared with other known proteins and if unique, thetechnique of degenerated PCR can be employed for obtaining the ligand.Once known, the affinity of the ligand will be determined by standardprotocols (15).

REFERENCES OF THE FIRST SERIES OF EXPERIMENTS

-   1. Chiaroda, A. (1991) National roundtable of prostate cancer:    research directions. Cancer Res. 51: 2498-2505.-   2. Coffey, D. S. Prostate Cancer—An overview of an increasing    dilemma. Cancer Supplement, 71,3: 880-886, 1993.-   3. Warner, J. A., et al., (1991) Future developments of non-hormonal    systemic therapy for prostatic carcinoma. Urologic Clin. North Amer.    18:25-33.-   4. Nguyen, L., et al., (1990) Prostatic acid phosphatase in the    serum of cancer patients with prostatic cancer is a specific    phosphotyrosine acid phosphatase. Clin. Chem. 35:1450-1455.-   5. Henttu, P., et al., (1989) cDNA coding for the entire human    prostate specific antigen show high homologies to the human tissue    kallikrein genes. Bioch. Biophys. Res. Comm. 160:903-908.-   6. Yong, CY-F., et al., (1991) Hormonal regulation of    prostate-specific antigen messenger RNA in human prostatic    adenocarcinoma cell line LNCaP. Cancer Res. 51:3748≅3752.-   7. Liotta, L. A. (1986) Tumor invasion and metastases: role of the    extracellular matrix. Cancer Res. 46:1-7.-   8. Horoszewicz, J. S., et al.. (1987) Monoclonal antibodies to a new    antigenic marker in epithelial prostatic cells and serum of    prostatic cancer patients. Anticancer Res. 7:927-936.-   9. Horoszewicz, J. S., et al. (1983) LNCaP model of human prostatic    carcinoma. Cancer Res., 43:1809-1818.-   10. Lopes, D., et al. (1990) Immunohistochemical and pharmacokinetic    characterization of the site-specific immunoconjugate CYT-356,    derived from anti-prostate monoclonal antibody 7E11-C5. Cancer Res.,    50:6423-6429.-   11. Wright, Jr., et al., (1990) Characterization of a new carcinoma    associated marker:7E11-C5. Antibod. Immunoconj.    Radiopharm.3:(abst#193).-   12. Feng, Q., et al., (1991) Purification and biochemical    characterization of the 7E11-C5 prostate carcinoma associated    antigen. Proc. Amer. Assoc. Cancer Res. 32:239.-   13. Axelrod, H. R., et al., Preclinical results and human    immunohistochemical studies with ⁹⁰Y-CYT-356. A New prostate cancer    agent. Abstract 596. AUA 87th Annual Meeting, May 10-14, 1992.    Washington, D.C.-   14. Maniatis, T., et al., (1982) Molecular Cloning; Cold Spring    Harbor Laboratory, pp.197-98 (1982).-   15. Maniatis, et al., (1982) Molecular Cloning, Cold Spring Harbor    Laboratory.-   16. Methods in Enzymology vol. 34: 1-810, 1974 (E) B. Jacoby and M.    Wilchek Academic Press, New York 1974.-   17. Hogan B. et al. (1986) Manipulating the Mouse Embryo, A    Laboratory Manual, Cold Spring Harbor Laboratory.-   18. Capecchi M. R. Science (1989) 244:1288-1292; Zimmer, A. and    Gruss, P. (1989) Nature 338:150-153.-   19. Trowbridge, I. S., (1982) Prospects for the clinical use of    cytotoxic monoclonal antibodies conjugates in the treatment of    cancer. Cancer Surveys 1:543-556.-   20. Hank, S. K. (1987) Homology probing: Identification of cDNA    clones encoding members of the protein-serine kinase family. Proc.    Natl. Acad. Sci. 84:368-392.-   21. Lee, C. C., et al., (1988) Generation of cDNA probes directed by    amino acid sequences: cloning of urate oxidase. Science, 239, 1288.-   22. Girgis, S. I., et al. (1988) Generation of DNA probes for    peptides with highly degenerate codons using mixed primer PCR.    Nucleic Acids Res. 16:10932.-   23. Kartner, N., et al. (1977) Isolation of plasma membranes from    human skin fibroblasts. J. Membrane Biology, 36:191-211.-   24. Hsu, S. M., et al. (1981) Comparative study of the    immunoperoxidase, anti-peroxidase, and avidin-biotin complex method    for studying polypeptide hormones with radioimmunoassay antibodies.    Am. J. Pathology, 75:734.-   25. Tempst, P., et al. (1989) Examination of automated polypeptide    sequencing using standard phenylisothiocyanate reagent and    subpicomole high performance liquid chromatography analysis.    Analytical Biochem. 183:290-300.-   26. Birnboim, H. C. (1983) A rapid alkaline extraction method for    the isolation of plasmid DNA. Meth. Enzymol, 100:243-255.-   27. Sanger, F., et al. (1977) DNA sequencing with chain-terminating    inhibitors. Proc. Natl. Acad. Sci. USA, 74:5463-5467.-   28. Grunstein, M., et al. (1975) Colony hybridization as a method    for the isolation of cloned DNAs that contain a specific gene. Proc.    Natl. Acad. Sci. USA, 72:3961.-   29. Feinberg, A. P., et al. (1983) A technique for radiolabeling DNA    restriction endonuclease fragments to high specific activity. Anal.    Biochem, 132, 6.-   30. Rave, N., et al. (1979) Identification of procollagen mRNAs    transferred to diazobenzylomethyl paper from formaldehyde gels.    Nucleic Acids Research, 6:3559.-   31. Voeller, H. J., et al. (1991) v-rasH expression confers    hormone-independent in-vitro growth to LNCaP prostate carcinoma    cells. Molec. Endocrinology. Vol. 5. No. 2, 209-216.-   32. Sirbasku, D. A. (1991) Purification of an equine apotransferrin    variant (thyromedin) essential for thyroid hormone dependent growth    of GH,, rat pituitary tumor cells in chemically defined culture.    Biochem.. 30:295-301.-   33. Rossi, M. C. (1992) Selective stimulation of prostatic carcinoma    cell proliferation by transferrin. Proc. Natl. Acad. Sci. (USA)    89:6197-6201.-   34. Eshhan, Z. (1990) Chimeric T cell receptor which incorporates    the anti-tumor specificity of a monoclonal antibody with the    cytolytic activity of T cells: a model system for immunotherapeutic    approach. B. J. Cancer 62:27-29.-   35. Antonie, P. (1990) Disposition of the prodrug 4-(bis(2    chloroethyl) amino)benzoyl-α-glutamic acid and its active parent in    mice. B. J. Cancer 62:905-914.-   36. Heimbrook, D. C.. et. al. (1990) Transforming growth factor    alpha-pseudomonas exotoxin fusion protein prolongs survival of nude    mice bearing tumor xenografts. Proc. Natl. Acad. Sci. (USA)    87:4697-4701.-   37. Chiarodo, A. National Cancer Institute roundtable on prostate    cancer, future research directions. Cancer Res., 51: 2498-2505,    1991.-   38. Abdel-Nabi, H., Wright, G. L., Gulfo, J. V, Petrylak, D. P.,    Neal, C. E., Texter, J. E., Begun, F. P., Tyson, I., Heal, A.,    Mitchell, E., Purnell, G., and Harwood, S. J. Monoclonal antibodies    and radioimmunoconjugates in the diagnosis and treatment of prostate    cancer. Semin. Urol., 10: 45-54, 1992.

SECOND SERIES OF EXPERIMENTS

Expression of the Prostate-Specific Membrane Antigen

Applicant's have recently cloned a 2.65 kb complementary DNA encodingPSM, the prostate-specific membrane antigen recognized by the 7E11-C5.3anti-prostate monoclonal antibody. Immunohistochemical analysis of theLNCaP, DU-145, and PC-3 prostate cancer cell lines for PSM expressionusing the 7E11-C5.3 antibody reveals intense staining in the LNCaPcells, with no detectable expression in both the DU-145 and PC-3 cells.Coupled in-vitro transcription/translation of the 2.65 kb full-lengthPSM cDNA yields an 84 kDa protein corresponding to the predictedpolypeptide molecular weight of PSM. Post-translational modification ofthis protein with pancreatic canine microsames yields the expected 100kDa PSM antigen. Following transfection of PC-3 cells with thefull-length PSM cDNA in a eukaryotic expression vector applicant'sdetect expression of the PSM glycoprotein by Western analysis using the7E11-C5.3 monoclonal antibody. Ribonuclease protection analysisdemonstrates that the expression of PSM mRNA is almost entirelyprostate-specific in human tissues. PSM expression appears to be highestin hormone-deprived states and is hormonally modulated by steroids, withDHT downregulating PSM expression in the human prostate cancer cell lineLNCaP by 8-10 fold, testosterone downregulating PSM by 3-4 fold, andcorticosteroids showing no significant effect. Normal and malignantprostatic tissues consistently show high PSM expression, whereas we havenoted heterogeneous, and at times absent, expression of PSM in benignprostatic hyperplasia. LNCaP tumors implanted and grown bothorthotopically and subcutaneously in nude mice, abundantly express PSMproviding an excellent in-vivo model system to study the regulation andmodulation of PSM expression.

EXPERIMENTAL DETAILS Materials and Methods

Cells and Reagents:

The LNCaP, DU-145, and PC-3 cell lines were obtained from the AmericanType Culture Collection. Details regarding the establishment andcharacteristics of these cell lines have been previously published(5A,7A,8A). Unless specified otherwise, LNCaP cells were grown in RPMI1640 media supplemented with L-glutamine, nonessential amino acids, and5% fetal calf serum (Gibco-BRL, Gaithersburg, Md.) in a CO₂ incubator at37C DU-145 and PC-3 cells were grown in minimal essential mediumsupplemented with 10% fetal calf serum. All cell media were obtainedfrom the MSKCC Media Preparation Facility. Restriction and modifyingenzymes were purchased from Gibco-BRL unless otherwise specified.

Immunohistochemical Detection of PSM

We employed the avidin-biotin method of detection to analyze prostatecancer cell lines for PSM antigen expression (9A). Cell cytospins weremade on glass slides using 5×10⁴ cells/100 ul per slide. Slides werewashed twice with PBS and then incubated with the appropriate suppressorserum for 20 minutes. The suppressor serum was drained off and the cellswere incubated with diluted 7E11-CS.3 (5 g/ml) monoclonal antibody for 1hour. Samples were then washed with PBS and sequentially incubated withsecondary antibodies for 30 minutes and with avidin-biotin complexes for30 minutes. Diaminobenzidine served as our chromogen and colordevelopment followed by hematoxylin counter-staining and mounting.Duplicate cell cytospins were used as controls for each experiment. As apositive control, the anti-cytokeratin monoclonal antibody CAM 5.2 wasused following the same procedure described above. Human EJ bladdercarcinoma cells served as a negative control.

In-Vitro Transcription/Translation of PSM Antigen

Plasmid 55A containing the full length 2.65 kb PSM cDNA in the plasmidpSPORT 1 (Gibco-BRL) was transcribed in-vitro using the Promega TNTsystem (Promega Corp. Madison, Wis.). T7 RNA polymerase was added to thecDNA in a reaction mixture containing rabbit reticulocyte lysate, anamino acid mixture lacking methionine, buffer, and ³⁵S-Methionine(Amersham) and incubated at 30C for 90 minutes. Post-translationalmodification of the resulting protein was accomplished by the additionof pancreatic canine microsomes into the reaction mixture (Promega Corp.Madison, Wis.). Protein products were analyzed by electrophoresis on 10%SDS-PAGE gels which were subsequently treated with Amplifyautoradiography enhancer (Amersham, Arlington Heights, Ill.) accordingto the manufacturers instructions and dried at 80C in a vacuum dryer.Gels were autoradiographed overnight at −70C using Hyperfilm MP(Amersham).

Transfection of PSM into PC-3 Cells

The full length PSM cDNA was subcloned into the pREP7 eukaryoticexpression vector (Invitrogen, San Diego, Calif.). Plasmid DNA waspurified from transformed DH5-alpha bacteria (Gibco-BRL) using Qiagenmaxi-prep plasmid isolation columns (Qiagen Inc., Chatsworth, Calif.).Purified plasmid DNA (6.10 g) was diluted with 900 ul of Optimem media(Gibco-BRL) and mixed with 30 ul of Lipofectin reagent (Gibco-BRL) whichhad been previously diluted with 9001 of Optimem media. This mixture wasadded to T-75 flasks of 40-50% confluent PC-3 cells in Optimem media.After 24-36 hours, cells were trypsinized and split into 100 mm dishescontaining RPMI 1640 media supplemented with 10% fetal calf serum and 1mg/ml of Hygromycin B (Calbiochem, La Jolla, Calif.). The dose ofHygromycin B used was previously determined by a time course/doseresponse cytotoxicity assay. Cells were maintained in this media for 2-3weeks with changes of media and Hygromycin B every 4-5 days untildiscrete colonies appeared. Colonies were isolated using 6 mm cloningcylinders and expanded in the same media. As a control, PC-3 cells werealso transfected with the pREP7 plasmid alone. RNA was isolated from thetransfected cells and PSM mRNA expression was detected by both RNaseProtection analysis (described later) and by Northern analysis.

Western Blot Detection of PSM Expression

Crude protein lysates, were isolated from LNCaP, PC-3, andPSM-transfected PC-3 cells as previously described (10A). LNCaP cellmembranes were also isolated according to published methods (10A).Protein concentrations were quantitated by the Bradford method using theBioRad protein reagent kit (BioRad, Richmond, Calif.). Followingdenaturation, 20 g of protein was electrophoresed on a 10% SDS-PAGE gelat 25 mA for 4 hours. Gels were electroblotted onto Immobilon Pmembranes (Millipore, Bedford, Mass.) overnight at 4C. Membranes wereblocked in 0.15M NaCl/0.01M Tris-HCl (TS) plus 5% BSA followed by a 1hour incubation with 7E11-C5.3 monoclonal antibody (10 g/ml). Blots werewashed 4 times with 0.15M NaCl/0.01M Tris-HCl/0.05% Triton-X 100 (TS-X)and incubated for 1 hour with rabbit anti-mouse IgG (AccurateScientific, Westbury, N.Y.) at a concentration of 10 g/ml.

Blots were then washed 4 times with TS-X and labeled with ¹²⁵I-Protein A(Amersham, Arlington Heights, Ill.) at a concentration of 1 millioncpm/ml. Blots were then washed 4 times with TS-X and dried on Whatman3MM paper, followed by overnight autoradiography at −70C using HyperfilmMP (Amersham).

Orthotopic and Subcutaneous LNCaP Tumor Growth in Nude Mice

LNCaP cells were harvested from sub-confluent cultures by a one minuteexposure to a solution of 0.25% trypsin and 0.02% EDTA. Cells wereresuspended in RPMI 1640 media with 5% fetal bovine scrum, washed anddiluted in either Matrigel (Collaborative Biomedical Products, Bedford,Mass.) or calcium and magnesium-free Hank's balanced salt solution(HBSS). Only single cell suspensions with greater than 90% viability bytrypan blue exclusion were used for in vivo injection. Male athymicSwiss (nu/nu) nude mice 4-6 weeks of age were obtained from the MemorialSloan-Kettering Cancer Center Animal Facility. For subcutaneous tumorcell injection one million LNCaP cells resuspended in 0.2 mils. ofMatrigel were injected into the hindlimb of each mouse using adisposable syringe fitted with a 28 gauge needle. For orthotopicinjection, mice were first anesthetized with an intraperitonealinjection of Pentobarbital and placed in the supine position. Theabdomen was cleansed with Betadine and the prostate was exposed througha midline incision. 2.5 million LNCaP tumor cells in 0.1 ml. wereinjected directly into either posterior lobe using a 1 ml disposablesyringe and a 28 gauge needle. LNCaP cells with and without Matrigelwere injected. Abdominal closure was achieved in one layer usingAutoclip wound clips (Clay Adams, Parsippany, N.J.). Tumors wereharvested in 6-8 weeks, confirmed histologically by faculty of theMemorial Sloan-Kettering Cancer Center Pathology Department, and frozenin liquid nitrogen for subsequent RNA isolation.

RNA Isolation

Total cellular RNA was isolated from cells and tissues by standardtechniques (11,12) as well as by using RNAzol B (Cinna/Biotecx, Houston,Tex.). RNA concentrations and quality were assessed by UV spectroscopyon a Beckman DU 640 spectrophotometer and by gel analysis. Human tissuetotal RNA samples were purchased from Clontech Laboratories, Inc., PaloAlto, Calif.

Ribonuclease Protection Assays

A portion of the PSM cDNA was subcloned into the plasmid vector pSPORT 1(Gibco-BRL) and the orientation of the cDNA insert relative to theflanking T7 and SP6 RNA polymerase promoters was verified by restrictionanalysis. Linearization of this plasmid upstream of the PSM insertfollowed by transcription with SP6 RNA polymerase yields a 400nucleotide antisense RNA probe, of which 350 nucleotides should beprotected from RNase digestion by PSM RNA. This probe was used in FIG.20. Plasmid IN-20, containing a 1 kb partial PSM cDNA in the plasmid pCRII (Invitrogen) was also used for riboprobe synthesis. IN-20 linearizedwith Xmn I (Gibco-BRL) yields a 298 nucleotide anti-sense RNA probe whentranscribed using SP6 RNA polymerase, of which 260 nucleotides should beprotected from RNase digestion by PSM mRNA. This probe was used in FIGS.21 and 22. Probes were synthesized using SP6 RNA polymerase (Gibco-BRL),rNTPs (Gibco-BRL) , RNAsin (Promega), and ³²P-rCTP (NEN, Wilmington,Del.) according to published protocols (13). Probes were purified overNENSORB 20 purification columns (NEN) and approximately 1 million cpm ofpurified, radiolabeled PSM probe was mixed with 10 g of each RNA andhybridized overnight at 45C using buffers and reagents from the RPA IIkit (Ambion, Austin, Tex.). Samples were processed as per manufacturer'sinstructions and analyzed on 5% polyacrilamide/7M urea denaturing gelsusing Seq ACRYL reagents (ISS, Natick, Mass.). Gels were pre-heated to55C and run for approximately 1-2 hours at 25 watts. Gels were thenfixed for 30 minutes in 10% methanol/10% acetic acid, dried onto Whatman3MM paper at 80C in a BioRad vacuum dryer and autoradiographed overnightwith Hyperfilm MP (Amersham)). Quantitation of PSM expression wasdetermined by using a scanning laser densitometer (LKB, Piscataway,N.J.).

Steroid Modulation Experiment

LNCaP cells (2 million) were plated onto T-75 flasks in RPMI 1640 mediasupplemented with 5% fetal calf serum and grown 24 hours untilapproximately 30-40% confluent. Flasks were then washed several timeswith phophate-buffered saline and RPMI medium supplemented with 5Tcharcoal-extracted scrum was added. Cells were then grown for another 24hours, at which time dihydrotesterone, testosterone, estradiol,progesterone, and dexamethasone (Steraloids Inc., Wilton, N.H.) wereadded at a final concentration of 2 nM. Cells were grown for another 24hours and RNA was then harvested as previously described and PSMexpression analyzed by ribonuclease protection analysis.

Experimental Results

Immunohistochemical Detection of PSM:

Using the 7E11-C5.3 anti-PSM monoclonal antibody, PSM expression isclearly detectable in the LNCaP prostate cancer cell line, but not inthe PC-3 and DU-145 cell lines (FIG. 17) in agreement with previouslypublished results (4A). All normal and malignant prostatic tissuesanalyzed stained positively for PSM expression (unpublished data).

In-Vitro Transcription/Translation of PSM Antigen:

As shown in FIG. 18, coupled in-vitro transcription/translation of the2.65 kb full-length PSM cDNA yields an 84 kDa protein species inagreement with the expected protein product from the 750 amino acid PSMopen reading frame. Following post-translational modification usingpancreatic canine microsomes we obtained a 100 kDa glycosylated proteinspecies consistent with the mature, native PSM antigen.

Detection of PSM Antigen in LNCaP Cell Membranes and Transfected PC-3Cells:

PC-3 cells transfected with the full length PSM cDNA in the pREP7expression vector were assayed for expression of SM mRNA by Northernanalysis (data not shown). A clone with high PSM mRNA expression wasselected for PSM antigen analysis by Western blotting using the7E11-C5.3 antibody. In FIG. 19, the 100 kDa PSM antigen is wellexpressed in LNCaP cell lysate and membrane fractions, as well as inPSM-transfected PC-3 cells but not in native PC-3 cells. This detectableexpression in the transfected PC-3 cells proves that the previouslycloned 2.65 kb PSM cDNA encodes the antigen recognized by the 7E11-C5.3anti-prostate monoclonal antibody and that the antigen is beingappropriately glycosylated in the PC-3 cells, since the antibodyrecognizes a carbohydrate-containing epitope on PSM.

PSH mRNA Expressions

Expression of PSM mRNA in normal human tissues was analyzed usingribonuclease protection assays. Tissue expression of PSM appearspredominantly within the prostate, with very low levels of expressiondetectable in human brain and salivary gland (FIG. 20). No detectablePSM mRNA expression was evident in non-prostatic human tissues whenanalyzed by Northern analysis (data not shown). We have also noted onoccasion detectable PSM expression in normal human small intestinetissue, however this mRNA expression is variable depending upon thespecific riboprobe used (data not shown). All samples of normal humanprostate and human prostatic adenocarcinoma assayed have revealedclearly detectable PSM expression, whereas we have noted generallydecreased or absent expression of PSM in tissues exhibiting benignhyperplasia (FIG. 21). In human LNCaP tumors grown both orthotopicallyand subcutaneously in nude mice we detected abundant PSM expression withor without the use of matrigel, which is required for the growth ofsubcutaneously implanted LNCaP cells (FIG. 21). PSM mRNA expression isdistinctly modulated by the presence of steroids in physiologic doses(FIG. 22). DHT downregulated expression by 8-10 fold after 24 hours andtestosterone diminished PSM expression by 3-4 fold. Estradiol andprogesterone also downregulated PSM expression in LNCaP cells, perhapsas a result of binding to the mutated androgen receptor known to existin the LNCaP cell. Overall, PSM expression is highest in the untreatedLNCaP cells grown in steroid-depleted media, a situation that we proposesimulates the hormone-deprived (castrate) state in-vivo. This experimentwas repeated at steroid dosages ranging from 2-200 nM and at time pointsfrom 6 hours to 7-days with similar results; maximal downregulation ofPSM mRNA was seen with DHT at 24 hours at doses of 2-20 nM.

Experimental Discussion

In order to better understand the biology of the human prostate in bothnormal and neoplastic states, we need to enhance our knowledge bystudying the various proteins and other features that are unique to thisimportant gland. Previous research has provided two valuable prostaticbiomarkers, PAP and PSA, both of which have had a significant impact onthe diagnosis, treatment, and management of prostate malignancies. Ourpresent work describing the preliminary characterization of theprostate-specific membrane antigen (PSM) reveals it to be a gene withmany interesting features. PSM is almost entirely prostate-specific asare PAP and PSA, and as such may enable further delineation of theunique functions and behavior of the prostate. The predicted sequence ofthe PSM protein (3) and its presence in the LNCaP cell membrane asdetermined by Western blotting and immunohistochemistry, indicate thatit is an integral membrane protein. Thus, PSM provides an attractivecell surface epitope for antibody-directed diagnostic imaging andcytotoxic targeting modalities (14). The ability to synthesize the PSMantigen in-vitro and to produce tumor xenografts maintaining high levelsof PSM expression provides us with a convenient and attractive modelsystem to further study and characterize the regulation and modulationof PSM expression. Also, the high level of PSM expression in the LNCaPcells provides an excellent in-vitro model system. Since PSM expressionis hormonally-responsive to steroids and may be highly expressed inhormone-refractory disease (15), it is imperative to elucidate thepotential role of PSM in the evolution of androgen-independent prostatecancer. The detection of PSM mRNA expression in minute quantities inbrain, salivary gland, and small intestine warrants furtherinvestigation, although these tissues were negative for expression ofPSM antigen by immunohistochemistry using the 7E11-C5.3 antibody (16).In all of these tissues, particularly small-intestine, we detected mRNAexpression using a probe corresponding to a region of the PSM cDNA nearthe 3′ end, whereas we were unable to detect expression when using a 5′end PSM probe. These results may indicate that the PSM mRNA transcriptundergoes alternative splicing in different tissues. Previous proteinstudies have suggested that the 7E11-C5.3 antibody may actually detecttwo other slightly larger protein species in addition to the 100 kDa PSMantigen (17). These other protein species can be seen in the LNCaPlysate and membrane samples in FIG. 19. Possible origins of theseproteins include alternatively spliced PSM mRNA, other genes distinctfrom but closely related to PSM, or different post-translationalmodifications of the PSM protein. We are currently investigating thesepossibilities.

Applicnat's approach is based on prostate tissue specificpromotor:enzyme or cytokine chimeras. We will examine promotor specificactivation of prodrugs such as non toxic gancyclovir which is convertedto a toxic metabolite by herpes simplex thymidine kinase or the prodrug4-(bis (2chloroethyl)amino)benzoyl-1-glutamic acid to the benzoic acidmustard alkylating agent by the pseudomonas carboxy peptidase G2. Asthese drugs are activated by the enzyme (chimera) specifically in thetumor the active drug is released only locally in the tumor environment,destroying the surrounding tumor cells. We will also examine thepromotor specific activation of cytokines such as IL-12, IL-2 or GM-CSFfor activation and specific antitumor vaccination. Lastly the tissuespecific promotor activation of cellular death genes may also prove tobe useful in this area.

Gene Therapy Chimeras

The establishment of “chimeric DNA” for gene therapy requires thejoining of different segments of DNA together to make a new DNA that hascharacteristics of both precursor DNA species involved in the linkage.In this proposal the two pieces being linked involve differentfunctional aspects of DNA, the promotor region which allows for thereading of the DNA for the formation of mRNA will provide specificityand the DNA sequence coding for the mRNA will provide for therapeuticfunctional DNA.

DNA-Specified Enzyme or Cytokine mRNA:

When effective, antitumor drugs can cause the regression of very largeamounts of tumor. The main requirements for antitumor drug activity isthe requirement to achieve both a long enough time (t) and high enoughconcentration (c) (c×t) of exposure of the tumor to the toxic drug toassure sufficient cell damage for cell death to occur. The drug alsomust be “active” and the toxicity for the tumor greater than for thehosts normal cells (22). The availability of the drug to the tumordepends on tumor blood flow and the drugs diffusion ability. Blood flowto the tumor does not provide for selectivity as blood flow to manynormal tissues is often as great or greater than that to the tumor. Themajority of chemotherapeutic cytotoxic drugs are often as toxic tonormal tissue as to tumor tissue. Dividing cells are often moresensitive than non-dividing normal cells, but in many slow growing solidtumors such as prostatic cancer this does not provide for antitumorspecificity (22).

Previously a means to increase tumor specificity of antitumor drugs wasto utilize tumor associated enzymes to activate nontoxic prodrugs tocytotoxic agents. (19). A problem with this approach was that most ofthe enzymes found in tumors were not totally specific in their activityand similar substrate active enzymes or the same enzyme at only slightlylower amounts was found in other tissue and thus normal tissues werestill at risk for damage.

To provide absolute specificity and unique activity, viral, bacterialand fungal enzymes which have unique specificity for selected prodrugswere found which were not present in human or other animal cells.Attempts to utilize enzymes such as herpes simplex thymidine kinase,bacterial cytosine deaminase and carboxypeptidase G-2 were linked toantibody targeting systems with modest success (19). Unfortunately,antibody targeted enzymes limit the number of enzymes available percell. Also, most antibodies do not have a high tumor target to normaltissue ratio thus normal tissues are still exposed reducing thespecificity of these unique enzymes. Antibodies are large molecules thathave poor diffusion properties and the addition of the enzymes molecularweight further reduces the antibodies diffusion.

Gene therapy could produce the best desired result if it could achievethe specific expression of a protein in the tumor and not normal tissuein order that a high local concentration of the enzyme be available forthe production in the tumor environment of active drug (21).

Cytokines:

Applicant's research group has demonstrated that Applicant's canspecifically and non-toxically “cure” an animal of an established tumor,in models of bladder or prostate cancer. The prostate cancer was themore difficult to cure especially if it was grown orthotopically in theprostate.

Our work demonstrated that tumors such as the bladder and prostate werenot immunogenic, that is the administration of irradiated tumor cells tothe animal prior to subsequent administration of non-irradiated tumorcells did not result in a reduction of either the number of tumor cellsto produce a tumor nor did it reduce the growth rate of the tumor. Butif the tumor was transfected with a retrovirus and secreted largeconcentrations of cytokines such as Il-2 then this could act as anantitumor vaccine and could also reduce the growth potential of analready established and growing tumor. IL-2 was the best, GM-CSF alsohad activity whereas a number of other cytokines were much less active.In clinical studies just using IL-2 for immunostimulation, very largeconcentrations had to be given which proved to be toxic. The key to thesuccess of the cytokine gene modified tumor cell is that the cytokine isproduced at the tumor site locally and is not toxic and that itstimulates immune recognition of the tumor and allows specific and nontoxic recognition and destruction of the tumor. The exact mechanisms ofhow IL-2 production by the tumor cell activates immune recognition isnot fully understood, but one explanation is that it bypasses the needfor cytokine production by helper T cells and directly stimulates tumorantigen activated cytotoxic CD8 cells. Activation of antigen presentingcells may also occur.

Tissue Promotor-Specific Chimera DNA Activation

Non-Prostatic Tumor Systems:

It has been observed in non-prostatic tumors that the use of promotorspecific activation can selectively lead to tissue specific geneexpression of the transfected gene. In melanoma the use of thetyrosinase promotor which codes for the enzyme responsible for melaninexpression produced over a 50 fold greater expression of the promotordriven reporter gene expression in melanoma cells and not non melanomacells. Similar specific activation was seen in the melanoma cellstransfected when they were growing in mice. In that experiment nonon-melanoma or melanocyte cell expressed the tyrosinase drive reportergene product. The research group at Welcome Laboratories have cloned andsequenced the promoter region of the gene -coding for carcinoembryonicantigen (CEA). CEA is expressed on colon and colon carcinoma cells butspecifically on metastatic cytosine deaminase which converts 5flurorocytosine into 5 fluorouracil and observed a large increase in theability to selectively kill CEA promotor driven colon tumor cells butnon dividing not dividing normal liver cells. In vivo they observed thatbystander tumor cells which were not transfected with the cytosinedeaminase gene were also killed, and that there was no toxicity to thehost animal as the large tumors were regressing following treatment.Herpes simplex virus, (HSV), thymidine kinase similarly activates theprodrug gancyclovir to be toxic towards dividing cancer cells and HSVthymidine kinase has been shown to be specifically activatable by tissuespecific promoters.

Prostatic Tumor Systems:

The therapeutic key to effective cancer therapy is to achievespecificity and spare the patient toxicity. Gene therapy may provide akey part to specificity in that non-essential tissues such as theprostate and prostatic tumors produce tissue specific proteins, such asacid phosphatase (PAP), prostate specific antigen (PSA), and a genewhich we cloned, prostate-specific membrane antigen (PSM). Tissues suchas the prostate contain selected tissue specific transcription factorswhich are responsible for binding to the promoter region of the DNA ofthese tissue specific mRNA. The promoter for PSA has been cloned and weare investigating its use as a prostate specific promotor for prostatictumor cells. Usually patients who are being treated for metastaticprostatic cancer have been put on androgen deprivation therapy whichdramatically reduces the expression of mRNA for PSA. PSM on the otherhand increases in expression with hormone deprivation which-means itwould be even more intensely expressed on patients being treated withhormone therapy. Preliminary work in collaboration with Dr. John Isaacs'Laboratory demonstrates that PSM is expressed when the human chromosomeregion containing the human PSM gene is transferred to the rat tumorAT-6. AT-6 is a metastatic androgen independent tumor. The samechromosome transferred into non prostate derived tissues or tumors isnot expressed and thus these cells could be used as an animal model forthese experiments. PSA, PSM positive Huan LNCaP cells will be used fortesting in nude mice.

REFERENCES OF THE SECOND SERIES OF EXPERIMENTS

-   1. Coffey, D. S. Prostate Cancer—An overview of an increasing    dilemma. Cancer Supplement, 71,3: 880-886, 1993.-   2. Chiarodo, A. National Cancer Institute roundtable on prostate    cancer; future research directions. Cancer Res., 51: 2498-2505,    1991.-   3. Israeli, R. S., Powell, C. T., Fair, W. R., and Heston, W. D. W.    Molecular cloning of a complementary DNA encoding a    prostate-specific membrane antigen. Cancer Res., 53: 227-230, 1993.-   4. Horoszewicz, J. S., Kawinski, E., and Murphy, G. P. Monoclonal    antibodies to a new antigenic marker in epithelial cells and serum    of prostatic cancer patients. Anticancer Res., 7: 927-936, 1987.-   5. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P.,    Rosenthal, H., Chu, T. M., Mirand, E. A., and Murphy, G. P. LNCaP    model of human prostatic carcinoma. Cancer Res., 43: 1809-1818,    1983.-   6. Abdel-Nabi, H., Wright, G. L., Gulfo, J. V., Petrylak, D. P.,    Neal, C. E., Texter, J. E., Begun, F. P., Tyson, I., Heal, A.,    Mitchell, E., Purnell, G.. and Harwood, S. J. Monoclonal antibodies    and radioimmunoconjugates in the diagnosis and treatment of prostate    cancer. Semin. Urol., 10: 45-54, 1992.-   7. Stone, K. R., Mickey, D. D., Wunderli, H.. Mickey, G. H., and    Paulson, D. F. Isolation of a human prostate carcinoma cell line    (DU-145). Int. J. Cancer, 22: 274-281, 1978.-   8. Kaign, M. E.. Narayan, K. S., Ohnuki, Y., and Lechner, J. F.    Establishment and characterization of a human prostatic carcinoma    cell line (PC-3). Invest. Urol., 17: 16-23, 1979.-   9. Hsu, S. M., Raine, L., and Panger, H. Review of present methods    of immunohistochemical detection. Am. J. Clin. Path. 75: 734-738,    1981.-   10. Harlow, E., and Lane, D. Antibodies: A Laboratory Manual. New    York: Cold Spring Harbor Laboratory, p. 449. 1968.-   11. Glisin, V, Crkvenjakov, R., and Byus, C. Ribonucleic acid    isolated by cesium chloride centrifugation. Biochemistry, 13:    2633-2637, 1974.-   12. Aviv, H., and Leder, P. Purification of biologically active    globin messenger RNA by chromotography on oligothymidylic acid    cellulose. Proc. Natl. Acad. Sci. USA, 69: 1408-1412, 1972.-   13. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T. A.,    Zinn, K., and Careen, M. R. Efficient in-vitro synthesis of    biologically active RNA and RNA hybridization probes from plasmids    containing a bacteriophage SP6 promoter. Nucl. Acids. Res. 12:    7035-7056, 1984.-   14. Personal Communication from Cytogen Corporation, Princeton, N.J.-   15. Axelrod, H. R., Gilman, S. C., D'Aleo, C. J., Petrylak, D.,    Reuter, V., Gulfo, J. V., Saad, A., Cordon-Cardo, C., and    Scher, H. I. Preclinical results and human immunohistochemical    studies with ⁹⁰Y-CYT-356; a new prostatic cancer therapeutic agent.    AUA Proceedings, Abstract 596, 1992.-   16. Lopes, A. D., Davis, W. L., Rosenstraus, M. J., Uveges, A. J.,    and Gilman, S. C. Immunohistochemical and pharmacokinetic    characterization of the site-specific immunoconjugate CYT-356    derived from antiprostate monoclonal antibody 7E11-C5. Cancer Res.,    50: 6423-6429, 1990.-   17. Troyer, J. K.. Qi, F., Beckett, M. L., Morningstar, M. M., and    Wright, G. L. Molecular characterization of the 7E11-CS prostate    tumor-associated antigen. AUA Proceedings. Abstract 482, 1993.-   18. Roemer, K., Friedmann, T. Concepts and strategies for human gene    therapy. FEBS. 223:212-225.-   19. Antonie, P. Springer, C. J., Bagshawe, F., Searle, F.,    Melton, R. G., Rogers, G. T., Burke, P. J., Sherwood, R. F.    Disposition of the prodrug 4-bis(2chloroethyl)amino)    benzoyl-1-glutamic acid and its active parent drug in mice.    Br.J.Cancer 62:909-914, 1990.-   20. Connor, J. Bannerji, R., Saito, S., Heston, W. D. W., Fair, W.    R., Gilboa, E. Regression of bladder tumors in mice treated with    interleukin 2 gene-modified tumor cells. J.Exp.Med.    177:1127-1134, 1993. (appendix)-   21. Vile R., Hart, I. R. In vitro and in vivo targeting of gene    expression to melanoma cells. Cancer Res. 53:962-967, 1993.-   22. Warner, J. A., Heston, W. D. W. Future developments of    nonhormonal systemic therapy for prostatic carcinoma. Urologic    Clinics of North America 18:25-33, 1991.-   23. Vile, R. G., Hart, I. R. Use of tissue specific expression of    the herpes simplex virus thymidine kinase gene to inhibit growth of    established murine melanomas following direct intratumoral injection    of DNA. Cancer Res. 53:3860-3864, 1993.

THIRD SERIES OF EXPERIMENTS Sensitive Detection of ProstaticHematogenous Micrometastases Using PSA and PSM-Derived Primers in thePolymerase Chain Reaction

We have developed a PCR-based assay enabling sensitive detection ofhematogenous micrometastases in patients with prostate cancer. Weperformed “nested PCR”, amplifying mRNA sequences unique toprostate-specific antigen and to the prostate-specific membrane antigen,and have compared their respective results. Micrametastases weredetected in 2/30 patients (6.7%) by PCR with PSA-derived primers, whilePSM-derived primers detected tumor cells in 19/16 patients (63.3%). All8 negative controls were negative with both PSA and PSM PCR. Assays wererepeated to confirm results, and PCR products were verified by DNAsequencing and Southern analysis. Patients harboring circulatingprostatic tumor cells as detected by PSM, and not by PSA-PCR included 4patients previously treated with radical prostatectomy and withnon-measurable serum PSA levels at the time of this assay. Thesignificance of these findings with respect to future disease recurrenceand progression will be investigated.

Improvement in the overall survival of patients with prostate cancerwill depend upon earlier diagnosis. Localized disease, without evidenceof extra-prostatic spread, is successfully treated with either radicalprostatectomy or external beam radiation, with excellent long-termresults (2,3). The major problem is that approximately two-thirds of mendiagnosed with prostate cancer already have evidence of advancedextra-prostatic spread at the time of diagnosis, for which there is atpresent no cure (4). The use of clinical serum markers such asprostate-specific antigen (PSA) and prostatic acid phosphatase (PAP)have enabled clinicians to detect prostatic carcinomas earlier andprovide useful parameters to follow responses to therapy (5). Yet,despite the advent of sensitive serum PSA assays, radionuclide bonescans, CT scans and other imaging modalities, we are still unable todetect the presence of micrometastatic cells prior to theirestablishment of solid metastases. Previous work has been done utilizingthe polymerase chain reaction to amplify mRNA sequences unique tobreast, leukemia, and other malignant cells in the circulation andenable early detection of micrometastases (6,7). Recently, a PCR-basedapproach utilizing primers derived from the PSA DNA sequence waspublished (8). In this study 3/12 patients with advanced, stage Dprostate cancer had detectable hematogenous micrometastases.

We have recently identified and cloned a 2.65 kb cDNA encoding the 100kDa prostate-specific membrane antigen (PSM) recognized by theanti-prostate monoclonal antibody 7E11-CS.3 (9). PSM appears to be anintegral membrane glycoprotein which is very highly expressed inprostatic tumors and metastases and is almost entirely prostate-specific(10). Many anaplastic tumors and bone metastases have variable and attimes no detectable expression of PSA, whereas these lesions appear toconsistently express high levels of PSM. Prostatic tumor cells thatescape from the prostate gland and enter the circulation are likely tohave the potential to form metastases and are possibly the moreaggressive and possibly anaplastic cells, a population of cells that maynot express high levels of PSA, but may retain high expression of PSM.We therefore chose to utilize DNA primers derived from the sequences ofboth PSA and PSM in a PCR assay to detect micrometastatic cells in theperipheral circulation. -Despite the high level of amplification andsensitivity of conventional RNA PCR, we have utilized a “nested” PCRapproach in which we first amplify a target sequence, and subsequentlyuse this PCR product as the template for another round of PCRamplification with a new set of primers totally contained within thesequence of the previous product. This approach has enabled us toincrease our level of detection from one prostatic tumor cell per 10,000cells to better than one cell per ten million cells.

EXPERIMENTAL DETAILS Materials and Methods

Cells and Reagents:

LNCaP and MCF-7 cells were obtained from the American Type CultureCollection (Rockville, Md.). Details regarding the establishment andcharacteristics of these cell lines have been previously published(11,12). Cells were grown in RPMI 1640 media supplemented withL-glutamine, nonessential amino acids, obtained from the MSKCC MediaPreparation Facility, and 5% fetal calf serum (Gibco-BRL, Gaithersburg,Md.) in a CO₂ incubator at 37C. All cell media was obtained from theMSKCC Media Preparation Facility. Routine chemical reagents were of thehighest grade possible and were obtained from Sigma Chemical Company,St. Louis, Mo.

Patient Blood Specimens

All blood specimens used in this study were from patients seen in theoutpatient offices of urologists on staff at MSKCC. Two anti-coagulated(purple top) tubes per patient were obtained at the time of theirregularly scheduled blood draws. Specimen procurement was conducted asper the approval of the MSKCC Institutional Review Board. Samples werepromptly brought to the laboratory for immediate processing. Serum PSAand PAP determinations were performed by standard techniques by theMSKCC Clinical Chemistry Laboratory. PSA determinations were performedusing the Tandem PSA assay (Hybritech, San Diego, Calif.). The eightblood specimens used as negative controls were from 2 males with normalscrum PSA values and biopsy-proven BPH, one healthy female, 3 healthymales, one patient with bladder cancer, and one patient with acutepromyelocytic leukemia.

Blood Sample Processing/RNA Extraction

4 ml of whole anticoagulated venous blood was mixed with 3 ml of icecold phosphate buffered saline and then carefully layered atop 8 ml ofFicoll (Pharmacia, Uppsala, Sweden) in a 15-ml polystyrene tube. Tubeswere centrifuged at 200 × g for 30 min. at 4C. Using a sterile pasteurpipette, the buffy coat layer (approx. 1 ml.) was carefully removed andrediluted up to 50 ml with ice cold phosphate buffered saline in a 50 mlpolypropylene tube. This tube was then centrifuged at 2000 × g for 30min at 4C. The supernatant was carefully decanted and the pellet wasallowed to drip dry. One ml of RNazol B was then added to the pellet andtotal RNA was isolated as per manufacturers directions (Cinna/Biotecx,Houston, Tex.). RNA concentrations and purity were determined by UVspectroscopy on a Beckman DU 640 spectrophotometer and by gelanalysis.

Determination of PCR Sensitivity

RNA was isolated from LNCaP cells and from mixtures of LNCaP and MCF-7cells at fixed ratios (i.e. 1:100, 1:1000, etc.) using RNAzol B. NestedPCR was then performed as described below with both PSA and PSM primersin order to determine the limit of detection for the assay. LNCaP:MCF-7(1:100,000) cDNA was diluted with distilled water to obtainconcentrations of 1:1.000,000 and 1:10,000,000. MCF-7 cells were chosenbecause they have been previously tested and shown not to express PSM byPCR.

Polymerase Chain Reaction

The PSA outer primers used span portions of exons 4 and 5 to yield a 486bp PCR product and enable differentiation between cDNA and possiblecontaminating genomic DNA amplification. The upstream primer sequencebeginning at nucleotide 494 in PSA cDNA sequence is5′-TACCCACTGCATCAGGAACA-3′ (SEQ. ID. No. 39) and the downstream primerat nucleotide 960 is 5′-CCTTGAAGCACACCATTACA-3′ (SEQ. ID. No. 40). ThePSA inner upstream primer (beginning at nucleotide 559)5′-ACACAGGCCAGGTATTTCAG-3′ (SEQ. ID. No. 41) and the downstream primer(at nucleotide 894) 5′-GTCCAGCGTCCAGCACACAG-3′ (SEQ. ID. No. 42) yield a355 bp PCR product. All primers were synthesized by the MSKCCMicrochemistry Core Facility. 5 g of total RNA was reverse-transcribedinto cDNA in a total volume of 201 using Superscript reversetranscriptase (Gibco-BRL) according to the manufacturersrecommendations. 11 of this cDNA served as the starting template for theouter primer PCR reaction. The 201 PCR mix included: 0.5U Taq polymerase(Promega Corp., Madison, Wis.), Promega reaction buffer, 1.5MM MgCl₂,200M dNTPs, and 1.0M of each primer. This mix was then transferred to aPerkin Elmer 9600 DNA thermal cycler and incubated for 25 cycles. ThePCR profile was as follows: 94C×15 sec., 60C×15 sec., and 72C for 45sec. After 25 cycles, samples were placed on ice, and 11 of thisreaction mix served as the template for another round of PCR using theinner primers. The first set of tubes were returned to the thermalcycler for 25 additional cycles. PSM-PCR required the selection ofprimer pairs that also spanned an intron in order to be certain thatcDNA and not genomic DNA were being amplified. Since the genomic DNAsequence of PSM has not yet been determined, this involved tryingdifferent primer pairs until a pair was found that produced the expectedsize PCR product when cDNA was amplified, but with no band produced froma genomic DNA template, indicating the presence of a large intron. ThePSM outer primers yield a 946 bp product and the inner primers a 434 bpproduct. The PSM outer upstream primer used was5′-ATGGTGTTTGTGGTATTACC-3′ (SEQ. ID. No. 43) (beginning at nucleotide1401) and the downstream primer (at nucleotide 2348) was5′-TGCTTGGAGCATAGATGACATGC-3′ (SEQ. ID. No. 44) The PSM inner upstreamprimer (at nucleotide 1581) was 5′-ACTCCTTCAAGAGCGTGGCG-3′ (SEQ. ID. No.45) and the downstream primer (at nucleotide 2015) was5′-AACACCATCCCTCCTCGAACC-3′ (SEQ. ID. No. 46). cDNA used was the same asfor the PSA assay. The 501 PCR mix included: 1U Tag Polymerase(Promega), 250M dNTPs, 10mM -mercaptoethanol, 2 mM MgCl₂, and 51 of a10× buffer mix containing: 166mM NH₄SO₄, 670mM Tris pH 8.8, and 2 mg/mlof acetylated BSA. PCR was carried out in a Perkin Elmer 480 DNA thermalcycler with the following parameters: 94C×4 minutes for 1 cycle, 94C×30sec., 58C×1 minute, and 72C×1 minute for 25 cycles, followed-by 72C×10minutes. Samples were then iced and 21 of this reaction mix was used asthe template for another 25 cycles with a new reaction mix containingthe inner PSM primers. cDNA quality was verified by performing controlreactions using primers derived from -actin yielding a 446 bp PCRproduct. The upstream primer used was 5′-AGGCCAACCGCGAGAAGATGA-3′ (SEQ.ID. No. 47) (exon 3) and the downstream primer was5′-ATGTCCACTGGGGAAGC-3′ (SEQ. ID. No. 48) (exon 4). The entire PSA mixand 101 of each PSM reaction mix were run on 1.5-2% agarose gels,stained with ethidium bromide and photographed in an Eagle Eye VideoImaging System (Stratagene, Torrey Pines, Calif.). Assays were repeatedat least 3 times to verify results.

Cloning and Sequencing of PCR Products

PCR products were cloned into the pCR II plasmid vector using the TAcloning system (Invitrogen). These plasmids were transformed intocompetent E. coli cells using standard methods (13) and plasmid DNA wasisolated using Magic Minipreps (Promega) and screened by restrictionanalysis. TA clones were then sequenced by the dideoxy method (14) usingSequenase (U.S. Biochemical). 3-4g of each plasmid was denatured withNaOH and ethanol precipitated. Labeling reactions were carried outaccording to the manufacturers recommendations using ³⁵S-dATP (NEN), andthe reactions were terminated as discussed in the same protocol.Sequencing products were then analyzed on 6% polyacrilamide/7M urea gelsrun at 120 watts for 2 hours. Gels were fixed for 20 minutes in 10%methanol/10% acetic acid, transferred to Whatman 3MM paper and drieddown in a vacuum dryer for 2 hours at 80C. Gels were thenautoradiographed at room temperature for 18 hours.

Southern Analysis

Ethidium-stained agarose gels of PCR products were soaked for 15 minutesin 0.2N HCl, followed by 30 minutes each in 0.5N NaOH/1.5M NaCl and 0.1MTris pH 7.5/1.5M NaCl. Gels were then equilibrated for 10 minutes in 10×SSC (1.5M NaCl/0.15M Sodium Citrate. DNA was transferred onto Nytrannylon membranes (Schleicher and Schuell) by pressure blotting in 10× SSCwith a Posi-blotter (Stratagene). DNA was cross-linked to the membraneusing a UV Stratalinker (Stratagene). Blots were pre-hybridized at 65Cfor 2 hours and subsequently hybridized with denatured ³²P-labeled,random-primed cDNA probes (either PSM or PSA) (9,15). Blots were washedtwice in 1× SSPE/0.5% SDS at 42C and twice in 0.1× SSPE/0.5% SDS at SOCfor 20 minutes each. Membranes were air-dried and autoradiographed for30 minutes to 1 hour at −70C with Kodak X-Omat film.

Experimental Results

Our technique of PCR amplification with nested primers improved ourlevel of detection of prostatic cells from approximately one prostaticcell per 10,000 MCF-7 cells to better than one cell per million MCF-7cells, using either PSA or PSM-derived primers (FIGS. 26 and 27). Thisrepresents a substantial improvement in our ability to detect minimaldisease. Characteristics of the 16 patients analyzed with respect totheir clinical stage, treatment, serum PSA and PAP values, and resultsof our assay are shown in table I. In total, PSA-PCR detected tumorcells in 2/30 patients (6.7%), whereas PSM-PCR detected cells in 19/30patients (63.3%). There were no patients positive for tumor cells by PSAand not by PSM, while PSM provided 8 positive patients not detected byPSA. Patients 10 and 11 in table 1, both with very advancedhormone-refractory disease were detected by both PSA and PSM. Both ofthese patients have died since the time these samples were obtained.Patients 4, 7, and 12, all of whom were treated with radicalprostatectomies for clinically localized disease, and all of whom havenon-measurable serum PSA values 1-2 years postoperatively were positivefor circulating prostatic tumor cells by PSM-PCR, but negative byPSA-PCR. A representative ethidium stained gel photograph for PSM-PCR isshown in FIG. 28. Samples run in lane A represent PCR products generatedfrom the outer primers and samples in lanes labeled B are products ofinner primer pairs. The corresponding PSM Southern blot autoradiographis shown in FIG. 29. The sensitivity of the Southern blot analysisexceeded that of ethidium staining, as can be seen in several sampleswhere the outer product is not visible on FIG. 28, but is detectable bySouthern blotting as shown in FIG. 29. In addition, sample 3 on FIGS. 28and 29 (patient 6 in FIG. 30) appears to contain both outer and innerbands that are smaller than the corresponding bands in the otherpatients. DNA sequencing has confirmed that the nucleotide sequence ofthese bands matches that of PSM, with the exception of a small deletion.This may represent either an artifact of PCR, alternative splicing ofPSM mRNA in this patient, or a PSM mutation. We have noted similarfindings with other samples on several occasions (unpublished data). Allsamples sequenced and analyzed by Southern analysis have been confirmedas true positives for PSA and PSM.

Experimental Details

The ability to accurately stage patients with prostate cancer at thetime of diagnosis is clearly of paramount importance in selectingappropriate therapy and in predicting long-term response to treatment,and potential cure. Pre-surgical staging presently consists of physicalexamination, serum PSA and PAP determinations, and numerous imagingmodalities including transrectal ultrasonography, CT scanning,radionuclide bone scans, and even MRI scanning. No present modality,however, addresses the issue of hematogenous micrometastatic disease andthe potential negative impact on prognosis that this may produce.Previous work has shown that only a fractional percentage of circulatingtumor cells will inevitably go on to form a solid metastasis (16),however, the detection of and potential quantification of circulatingtumor cell burden may prove valuable in more accurately staging disease.The long-term impact of hematogenous micrometastatic disease must bestudied by comparing the clinical courses of patients found to havethese cells in their circulation with patients of similar stage andtreatment who test negatively.

The significantly higher level of detection of tumor cells with PSM ascompared to PSA is not surprising to us, since we have noted moreconsistent expression of PSM in prostate carcinomas of all stages andgrades as compared to variable expression of PSA in more poorlydifferentiated and anaplastic prostate cancers. We were surprised todetect tumor cells in the three patients that had undergone radicalprostatectomies with subsequent undetectable amounts of serum PSA. Thesepatients would be considered to be surgical “cures” by standardcriteria, yet they apparently continue to harbor prostatic tumor cells.It will be interesting to follow the clinical course of these patientsas compared to others without PCR evidence of residual disease. We arepresently analyzing larger numbers of patient samples in order to verifythese findings and perhaps identify patients at risk for metastaticdisease.

REFERENCES

-   1. Boring, C. C., Squires, T. S., and Tong, T.: Cancer    Statistics, 1993. CA Cancer J. Clin., 43:7-26, 1993.-   2. Lepor, H., and Walsh, P. C.: Long-term results of radical    prostatectomy in clinically localized prostate cancer: Experience at    the Johns Hopkins Hospital. NCI Monogr., 7:117-122, 1988.-   3. Bagshaw, M. A., Cox, R. S., and Ray, G. R.: Status of radiation    treatment of prostate cancer at Stanford University. NCI Monogr.,    7:47-60, 1988.-   4. Thompson, I. M., Rounder, J. B., Teague, J. L., et al.: Impact of    routine screening for adenocarcinoma of the prostate on stage    distribution. J. Urol., 137:424-426, 1987.-   5. Chiarodo, A.: A National Cancer Institute roundtable on prostate    cancer, future-research directions. Cancer Res., 51:2498-2505, 1991.-   6. Wu, A., Ben-Ezra, J., and Colombero, A.: Detection of    micrometastasis in breast cancer by the polymerase chain reaction.    Lab. Invest., 62:109A, 1990.-   7. Fey, M. F.. Kulozik, A. E., and Hansen-Hagge, T. E.: The    polymerase chain reaction: A new tool for the detection of minimal    residual disease in hematological malignancies. Eur. J. Cancer,    27:89-94, 1991.-   8. Moreno, J. G., Croce, C. M., Fischer, R., Monne, M., Vihko, P.,    Mulholland, S. G., and Gomella, L. G.: Detection of hematogenous    micrometastasis in patients with prostate cancer. Cancer Res.,    52:6110-6112, 1992.-   9. Israeli, R. S., Powell, C. T., Fair, W. R., and Heston, W. D. W.:    Molecular cloning of a complementary DNA encoding a    prostate-specific membrane antigen. Cancer Res., 53:227-230, 1993.-   10. Israeli, R. S., Powell, C. T., Corr, J. G., Fair, W. R., and    Heston, W. D. W.: Expression of the prostate-specific membrane    antigen (PSM).: Submitted to Cancer Research.-   11. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P.,    Rosenthal, H., Chu. T. M., Mirand, E. A., and Murphy, G. R: LNCaP    model of human prostatic carcinoma. Cancer Res., 43:1809-1818, 1983.-   12. Soule, H. D., Vazquez, J., Long, A., Albert, S., and Brennan,    M.: A human cell line from a pleural effusion derived from a breast    carcinoma. J. Natl. Can. Inst., 51:1409-1416, 1973.-   13. Hanahan, D.: Studies on transformation of Escherichia coli with    plasmids. J. Mol. Biol., 166:557-580, 1983.-   14. Sanger, F., Nicklen, S., and Coulson, A. R.: DNA sequencing with    chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA,    74:5463-5467, 1977.-   15. Lundwall, A., and Lilja, H.: Molecular cloning of a human    prostate specific antigen cDNA. FEBS Letters, 214:317, 1987.-   16. Liotta, L. A., Kleinerman, J., and Saidel, G. M.; Quantitative    relationships of intravascular tumor cells, tumor vessels, and    pulmonary metastases following tumor implantation. Cancer Res.,    34:997-1003, 1974.

FOURTH SERIES OF EXPERIMENTS EXPRESSION OF THE PROSTATE SPECIFICMEMBRANE ANTIGEN (PSM) DIMINISHES THE MITOGENIC STIMULATION OFAGGRESSIVE HUMAN PROSTATIC CARCINOMA CELLS BY TRANSFERRIN

An association between transferrin and human prostate cancer has beensuggested by several investigators. It has been shown that the expressedprostatic secretions of patients with prostate cancer are enriched withrespect to their content of transferrin and that prostate cancer cellsare rich in transferrin receptors (J. Urol. 143, 381, 1990). Transferrinderived from bone marrow has been shown to selectively stimulate thegrowth of aggressive prostate cancer cells (PNAS 89, 6197, 1992). Wehave previously reported the cloning of the cDNA encoding the 100 kDaPSM antigen (Cancer Res. 53, 208, 1993). DNA sequence analysis hasrevealed that a portion of the coding region, from nucleotide 1250 to1700 possesses a 54% homology to the human transferrin receptor. PC-3cells do not express PSM mRNA or protein and exhibit increased cellgrowth in response to transferrin, whereas, LNCaP prostate cancer cellswhich highly express PSM have a very weak response to transferrin. Todetermine whether PSM expression by prostatic cancer cells impacts upontheir mitogenic response to transferrin we stably transfected thefull-length PSM cDNA into the PC-3 prostate cancer cells. Clones highlyexpressing PSM mRNA were identified by Northern analysis and expressionof PSM protein was verified by Western analysis using the anti-PSMmonoclonal antibody 7E11-C5.3.

We plated 2×10⁴ PC-3 or PSM-transfected PC-3 cells per well in RPMImedium supplemented with 10% fetal bovine serum and at 24 hrs. added 1μg per ml. of holotransferrin to the cells. Cells were counted at 1 dayto be highly mitogenic to the PC-3 cells. Cells were counted at 1 day todetermine plating efficiency and at 5 days to determine the effect ofthe transferrin. Experiments were repeated to verify the results.

We found that the PC-3 cells experienced an average increase of 275%over controls, whereas the LNCaP cells were only stimulated 43%. Growthkinetics revealed that the PSM-transfected PC-3 cells grew 30% slowerthan native PC-3 cells. This data suggests that PSM expression inaggressive, metastatic human prostate cancer cells significantlyabrogates their mitogenic response to transferrin.

The use of therapeutic vaccines consisting of cytokine-secreting tumorcell preparations for the treatment cf established prostate cancer wasinvestigated in the Dunning R3327-MatLyLu rat prostatic adenocarcinomamodel. Only IL-2 secreting, irradiated tumor cell preparations werecapable of curing animals from subcutaneously established tumors, andengendered immunological memory that protected the animals from anothertumor challenge. Immunotherapy was less effective when tumors wereinduced orthotopically, but nevertheless led to improved outcome,significantly delaying, and occasionally preventing recurrence of tumorsafter resection of the cancerous prostate. Induction of a potent immuneresponse in tumor bearing animals against the nonimmunogenic MatLyLutumor supports the view that active immunotherapy of prostate cancer mayhave therapeutic benefits.

1. A purified monoclonal antibody which binds to a fragment of an outermembrane domain of prostate specific membrane antigen, which fragmenthas within its structure the consecutive amino acid sequenceAsp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO: 35).
 2. A purified monoclonalantibody which binds to a fragment of an outer membrane domain ofprostate specific membrane antigen, which fragment has within itsstructure the consecutive amino acid sequence Asn-Glu-Asp-Gly-Asn-Glu(SEQ ID NO: 36).
 3. A purified monoclonal antibody which binds to afragment of an outer membrane domain of prostate specific membraneantigen, which fragment has within its structure the consecutive aminoacid sequence Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO: 37).
 4. A purifiedmonoclonal antibody which binds to a fragment of an outer membranedomain of prostate specific membrane antigen, which fragment has withinits structure each of the following amino acid sequences: (a)Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO: 35); (b) Asn-Glu-Asp-Gly-Asn-Glu(SEQ ID NO: 36); (c) Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO: 37); and (d)Ala-Gly-Ala-Leu-Val-Leu-Ala-Gly-Gly-Phe-Phe-Leu-Leu-Gly-Phe-Leu-Phe (SEQID 80:38).
 5. A purified monoclonal antibody which binds to a fragmentof an outer membrane domain of prostate specific membrane antigen, whichfragment has within its structure each of the following amino acidsequences: (a) Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO: 35); (b)Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO: 36); and (c) Lys-Ser-Pro-Asp-Glu-Gly(SEQ ID NO: 37).
 6. A purified monoclonal antibody which binds to afragment of prostate specific membrane antigen, which fragmentcorresponds to a hydrophilic region of an outer membrane domain ofprostate specific membrane antigen, the amino acid sequence of whichantigen is set forth in SEQ ID NO:2.
 7. A purified monoclonal antibodywhich binds to a hydrophilic region of an outer membrane domain ofprostate specific membrane antigen, which hydrophilic region has withinits structure the consecutive amino acid sequenceAsp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO: 35).
 8. A purified monoclonalantibody which binds to a hydrophilic region of an outer membrane domainof prostate specific membrane antigen, which hydrophilic region haswithin its structure the consecutive amino acid sequenceAsn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO: 36).
 9. A purified monoclonalantibody which binds to a hydrophilic region of an outer membrane domainof prostate specific membrane antigen, which hydrophilic region haswithin its structure the consecutive amino acid sequenceLys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO: 37).
 10. A purified monoclonalantibody which binds to an outer membrane domain of prostate specificmembrane antigen, the amino acid sequence of which antigen is set forthin SEQ ID NO:2.
 11. A purified monoclonal antibody which binds to ahydrophilic region of an outer membrane domain of prostate specificmembrane antigen, the amino acid sequence of which antigen is set forthin SEQ ID NO:2.
 12. The purified antibody of any one of claims 1-11,wherein the antibody is a monoclonal antibody.
 13. A composition ofmatter comprising the a monoclonal antibody of any one of 1-11 and anagent conjugated to the monoclonal antibody thereto, wherein themonoclonal antibody binds to an outer membrane domain ofprostate-specific membrane antigen, the amino acid sequence of whichantigen is set forth in SEQ ID NO:2.
 14. The composition of matter ofclaim 13, wherein the agent is a radioisotope or toxin.
 15. Acomposition comprising a carrier and the composition of matter of claim13.
 16. A method of imaging prostate cancer in a subject which comprisesadministering to the subject the composition of matter of claim 13,wherein the agent is an imaging agent under conditions permittingformation of a complex between the composition of matter and prostatespecific membrane antigen, and obtaining an image of any complex soformed.
 17. A monoclonal antibody having an antigen-binding region-specific for the extracellular domain of prostate specific membraneantigen, the amino acid sequence of which antigen is set forth in SEQ IDNO:2.
 18. A composition of matter comprising a monoclonal antibody andan agent conjugated thereto, wherein the monoclonal antibody binds to afragment of an outer membrane domain of prostate-specific membraneantigen, which fragment has within its structure the consecutive aminoacid sequence Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO:35).
 19. A compositionof matter comprising a monoclonal antibody and an agent conjugatedthereto, wherein the monoclonal antibody binds to a fragment of an outermembrane domain of prostate-specific membrane antigen, which fragmenthas within its structure the consecutive amino acid sequenceAsn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO:36).
 20. A composition of mattercomprising a monoclonal antibody and an agent conjugated thereto,wherein the monoclonal antibody binds to a fragment of an outer membranedomain of prostate-specific membrane antigen, which fragment has withinits structure the consecutive amino acid sequenceLys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO:37).
 21. A composition of mattercomprising a monoclonal antibody and an agent conjugated thereto,wherein the monoclonal antibody binds to a fragment of an outer membranedomain of prostate-specific membrane antigen, which fragment has withinits structure each of the following amino acid sequences: (a)Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO: 35); (b) Asn-Glu-Asp-Gly-Asn-Glu(SEQ ID NO: 36); and (c) Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO: 37).
 22. Acomposition of matter comprising a monoclonal antibody and an agentconjugated thereto, wherein the monoclonal antibody binds to a fragmentof prostate-specific membrane antigen, which fragment corresponds to ahydrophilic region of an outer membrane domain of prostate-specificmembrane antigen, the amino acid sequence of which antigen is set forthin SEQ ID NO:2.
 23. A composition of matter comprising a monoclonalantibody and an agent conjugated thereto, wherein the monoclonalantibody binds to a hydrophilic region of an outer membrane domain ofprostate-specific membrane antigen, which hydrophilic region has withinits structure the consecutive amino acid sequenceAsp-Glu-Leu-Lys-Ala-Glu (SEQ ID NO:35).
 24. A composition of mattercomprising a monoclonal antibody and an agent conjugated thereto,wherein the monoclonal antibody binds to a hydrophilic region of anouter membrane domain of prostate-specific membrane antigen, whichhydrophilic region has within its structure the consecutive amino acidsequence Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO:36).
 25. A composition ofcomprising a monoclonal antibody and an agent conjugated thereto,wherein the monoclonal antibody binds to a hydrophilic region of anouter membrane domain of prostate-specific membrane antigen, whichhydrophilic region has within its structure the consecutive amino acidsequence Lys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO:37).
 26. A composition ofmatter comprising a monoclonal antibody and an agent conjugated thereto,wherein the monoclonal antibody binds to a hydrophilic region of anouter membrane domain of prostate specific membrane antigen, the aminoacid sequence of which antigen is set forth in SEQ ID NO:2.
 27. Thecomposition of matter of any of claim 18-20, or 21-26, wherein the agentis a radioisotope or toxin.
 28. A composition comprising a carrier andthe composition of matter of any of claim 18-20 or 21-26.
 29. A methodof imaging prostate cancer in a subject which comprises administering tothe subject the composition of matter of any of claim 18-20 or 21-26,wherein the agent is an imaging agent, under conditions permittingformation of a complex between the composition of matter andprostate-specific membrane antigen, and obtaining an image of anycomplex so formed.
 30. A composition comprising a carrier and thecomposition of matter of claim
 27. 31. The composition of claim 30,wherein the agent is a radioisotope.
 32. The composition of claim 30,wherein the agent is a toxin.
 33. The method of claim 29, wherein theimaging agent is a radioisotope.